Programmable illumination pattern for transporting microparticles

ABSTRACT

An apparatus providing programmable illumination pattern generation for the manipulation of colloidal particulates and biomolecules in suspension between electrodes is disclosed. The apparatus implements LEAPS (light-controlled electrokinetic assembly of particles near surfaces), which relies on AC electric field-induced assembly of particles, patterning of an electrolyte/silicon oxide/silicon interface to exert spatial control over the assembly process, and real-time control of assembly via external illumination. The apparatus generates patterns of illumination and projects them onto planar surfaces, i.e., LEAPS electrodes. This enables the creation of patterns using graphical design or drawing software on a personal computer and the projection of said patterns or sequences of patterns (“time-varying patterns”) onto the interface using a liquid crystal display (LCD) panel and an optical design that images the LCD panel onto the surface of interest to provide for the assembly of particles within such patterns.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/624,020, filed on Jul. 21, 2003, now U.S. Pat. No. 7,390,676, whichis a continuation of U.S. application Ser. No. 09/768,414, filed Jan.24, 2001, now U.S. Pat. No. 7,041,510, which is a continuation of U.S.application Ser. No. 09/397,793, filed Sep. 17, 1999, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 09/171,550,filed Oct. 26, 1998, now U.S. Pat. No. 6,251,691, which is a nationalphase application of PCT/US97/08159, filed Apr. 24, 1997, which claimspriority to provisional application 60/016,642, filed Apr. 25, 1996.Each of these applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention generally relates to the field of materialsscience and analytical chemistry.

The present invention specifically relates to the realization of acomplete, functionally integrated system for the implementation ofbiochemical analysis in a planar, miniaturized format on the surface ofa conductive and/or photoconductive substrate, with applications inpharmaceutical and agricultural drug discovery and in in-vitro orgenomic diagnostics. In addition, the method and apparatus of thepresent invention may be used to create material surfaces exhibitingdesirable topographical relief and chemical functionality, and tofabricate surface-mounted optical elements such as lens arrays.

BACKGROUND OF THE INVENTION

I—Ions, Electric Fields and Fluid Flow: Field-induced Formation ofPlanar Bead Arrays

Electrokinesis refers to a class of phenomena elicited by the action ofan electric field on the mobile ions surrounding charged objects in anelectrolyte solution. When an object of given surface charge is immersedin a solution containing ions, a diffuse ion cloud forms to screen theobject's surface charge. This arrangement of a layer of (immobile)charges associated with an immersed object and the screening cloud of(mobile) counter-ions in solution is referred to as a “double layer” Inthis region of small but finite thickness, the fluid is notelectroneutral. Consequently, electric fields acting on this region willset in motion ions in the diffuse layer, and these will in turn entrainthe surrounding fluid. The resulting flow fields reflect the spatialdistribution of ionic current in the fluid Electroosmosis represents thesimplest example of an electrokinetic phenomenon. It arises when anelectric field is applied parallel to the surface of a sample containeror electrode exhibiting fixed surface charges, as in the case of asilicon oxide electrode (in the range of neutral pH). As counter-ions inthe electrode double layer are accelerated by the electric field, theydrag along solvent molecules and set up bulk fluid flow. This effect canbe very substantial in narrow capillaries and may be used to advantageto devise fluid pumping systems.

Electrophoresis is a related phenomenon which refers to thefield-induced transport of charged particles immersed in an electrolyte.As with electroosmosis, an electric field accelerates mobile ions in thedouble layer of the particle, if, in contrast to the earlier case, theparticle itself is mobile, it will compensate for this field-inducedmotion of ions (and the resulting ionic current) by moving in theopposite direction. Electrophoresis plays an important role inindustrial coating processes and, along with electroosmosis, it is ofparticular interest in connection with the development of capillaryelectrophoresis into a mainstay of modern bioanalytical separationtechnology.

In confined geometries, such as that of a shallow experimental chamberin the form of a “sandwich” of two planar electrodes, the surface chargedistribution and topography of the bounding electrode surfaces play aparticularly important role in determining the nature and spatialstructure of electroosmotic flow. Such a “sandwich” electrochemical cellmay be formed by a pair of electrodes separated by a shallow gap.Typically, the bottom electrode to will be formed by an oxide-cappedsilicon wafer while the other electrode is formed by opticallytransparent conducting indium tin oxide (ITO). The silicon (Si) waferrepresents a thin slice of a single crystal of silicon which is doped toattain suitable levels of electrical conductivity and insulated from theelectrolyte solution by a thin layer of silicon oxide (SiOx).

The reversible aggregation of beads into planar aggregates adjacent toan electrode surface may be induced by a (DC or AC) electric field thatis applied normal to the electrode surface. While the phenomenon hasbeen previously observed in a cell formed by a pair of conductive ITOelectrodes (Richetti, Prost and Barois, J. Physique Lettr. 45, L-1137through L-1143 (1984)), the contents of which are incorporated herein byreference, it has been only recently demonstrated that the underlyingattractive interaction between beads is mediated by electrokinetic flow(Yeh, Seul and Shraiman, “Assembly of Ordered Colloidal Aggregates byElectric Field Induced Fluid Flow”, Nature 386, 57-59 (1997), thecontents of which are incorporated herein by reference). This flowreflects the action of lateral non-uniformities in the spatialdistribution of the current in the vicinity of the electrode. In thesimplest case, such non-uniformities are introduced by the very presenceof a colloidal bead near the electrode as a result of the fact that eachbead interferes with the motion of ions in the electrolyte. Thus, it hasbeen observed that an individual head, when placed near the electrodesurface, generates a toroidal flow of fluid centered on the bead.Spatial non-uniformities in the properties of the electrode can also beintroduced deliberately by several methods to produce lateral fluid flowtoward regions of low impedance. These methods are described insubsequent sections below.

Particles embedded in the electrokinetic flow are advected regardless oftheir specific chemical or biological nature, while simultaneouslyaltering the flow field. As a result, the electric field-inducedassembly of planar aggregates and arrays applies to diverse colloidalparticles including: beaded polymer resins (“beads”), lipid vesicles,whole chromosomes, cells and biomolecules including proteins and DNA, aswell as metal or semiconductor colloids and clusters.

Important for the applications to be described is the fact that theflow-mediated attractive interaction between beads extends to distancesfar exceeding the characteristic bead dimension. Planar aggregates areformed in response to an externally applied electric field anddisassemble when the field is removed. The strength of the applied fielddetermines the strength of the attractive interaction that underlies thearray assembly process and thereby selects the specific arrangementadopted by the beads within the array. That is, as a function ofincreasing applied voltage, beads first form planar aggregates in whichparticles are mobile and loosely packed, then assume a tighter packing,and finally exhibit a spatial arrangement in the form of a crystalline,or ordered, array resembling a raft of bubbles. The sequence oftransitions between states of increasing internal order is reversible,including complete disassembly of planar aggregates when the appliedvoltage is removed. In another arrangement, at low initialconcentration, beads form small clusters which in turn assume positionswithin an ordered “superstructure”.

II—Pattering of Silicon Oxide Electrode Surfaces

Electrode patterning in accordance with a predetermined designfacilitates the quasi-permanent modification of the electrical impedanceof the EIS (Electrolyte-Insulator Semiconductor) structure of interesthere. By spatially modulating the EIS impedance, electrode-patterningdetermines the ionic current in the vicinity of the electrode. Dependingon the frequency of the applied electric field, beads either seek out,or avoid, regions of high ionic current. Spatial patterning thereforeconveys explicit external control over the placement and shape of beadarrays.

While patterning may be achieved in many ways, two procedures offerparticular advantages. First, UV-mediated re-growth of a thin oxidelayer on a properly prepared silicon surface is a convenient methodologythat avoids photolithographic resist patterning and etching. In thepresence of oxygen, UV illumination mediates the conversion of exposedsilicon into oxide. Specifically, the thickness of the oxide layerdepends on the exposure time and may thus be spatially modulated byplacing patterned masks into the UV illumination path. This modulationin thickness, with typical variations of approximately 10 Angstroms,translates into spatial modulations in the impedance of the Si/SiOxinterface while leaving a flat and chemically homogeneous top surfaceexposed to the electrolyte solution. Send spatial modulations in thedistribution of the electrode surface charge may be produced byUV-mediated photochemical oxidation of a suitable chemical species thatis first deposited as a monolayer film on the SiOx surface. This methodpermits fine control over local features of the electrode double layerand thus over the electrokinetic flow.

A variation of this photochemical modulation is the creation of lateralgradients in the EIS impedance and hence in the current generated inresponse to the applied electric field. For example, this is readilyaccomplished by controlling the UV exposure so as to introduce a slowlateral variation in the oxide thickness or in the surface chargedensity. As discussed below, control over lateral gradients serves toinduce lateral bead transport and facilitates the implementation of suchfundamental operations as capturing and channeling of beads to apredetermined destination along conduits in the form of impedancefeatures embedded in the Si/SiOx interface Photochemical patterning offunctionalized chemical overlayers also applies to other types ofelectrode surfaces including ITO.

III—Light-Controlled Modulation of the Interfacial Impedance

The spatial and temporal modulation of the EIS-impedance in accordancewith a pattern of external illumination provides the basis to controlthe electrokinetic forces that mediate bead aggregation. Thelight-modulated electrokinetic assembly of planar colloidal arraysfacilitates remote interactive control over the formation, placement andrearrangement of bead arrays in response to corresponding illuminationpatterns and thereby offers a wide range of interactive manipulations ofcolloidal beads and biomolecules.

To understand the principle of this methodology, it will be helpful tobriefly review pertinent photoelectric properties of semiconductors, ormore specifically, those of the EIS structure formed by the Electrolytesolution (F), the Insulating SiOx layer (I) and the Semiconductor (S).The photoelectric characteristics of this structure are closely relatedto those of a standard Metal-Insulator-Semiconductor (MIS) orMetal-Oxide-Semiconductor (MOS) devices which are described in S. M.Sze, “The Physics of Semiconductors”. 2nd Edition, Chapt. 7 (WileyInterscience 1981), the contents of which are incorporated herein byreference.

The interface between the semiconductor and the insulating oxide layerdeserves special attention. Crucial to the understanding of theelectrical response of the MOS stricture to light is the concept of aspace charge region of small but finite thickness that forms at theSi/SiOx interface in the presence of a bias potential. In the case ofthe EIS structure an effective bias, in the form of a junctionpotential, is present under all but very special conditions. The spacecharge region forms in response to the distortion of the semiconductor'svalence and conduction bands (“band bending”) in the vicinity of theinterface. This condition in turn reflects the fact that, while there isa bias potential across the interface, there is ideally no chargetransfer in the presence of the insulating oxide. That is, inelectrochemical language, the EIS structure eliminates Faradaic effects.Instead, charges of opposite sign accumulate on either side of theinsulating oxide layer and generate a finite polarization.

In the presence of a reverse bias, the valence and conduction band edgesof an n-doped semiconductor bend upward near the Si/SiOx interface andelectrons flow out of the interfacial region in response to thecorresponding potential gradient. As a result a majority carrierdepletion layer is formed in the vicinity of the Si/SiOx interface.Light absorption in the semiconductor provides a mechanism to createelectron-hole pairs within this region. Provided that they do notinstantaneously recombine, electron-hole pairs are split by the locallyacting electric field, and a corresponding photocurrent flows. It isthis latter effect that affords control over the electrokinetic assemblyof beads in the electrolyte solution.

To understand in more detail the pertinent frequency dependence of thelight-induced modulation of the EIS impedance, two aspects of theequivalent circuit representing the EIS structure are noteworthy. First,there are close analogies between the detailed electricalcharacteristics of the electric double layer at the electrolyte-oxideinterface, and the depletion layer at the interface between thesemiconductor and the insulator. As with the double layer, the depletionlayer exhibits electrical characteristics similar to those of acapacitor with a voltage-dependent capacitance. As discussed,illumination serves to lower the impedance of the depletion layer.Second, given its capacitive electrical response, the oxide layer willpass current only above a characteristic (“threshold”) frequency.Consequently, provided that the frequency of the applied voltage exceedsthe threshold, illumination can lower the effective impedance of theentire EIS structure.

This effective reduction of the EIS impedance also depends on the lightintensity which determines the rate of generation of electron-holepairs. In the absence of significant recombination, the majority ofphotogenerated electrons flow out of the depletion region and contributeto the photocurrent. The remaining hole charge accumulates near theSi/SiOx interface and screens the electric field acting in the depletionregion. As a result, the rate of recombination increases, and theefficiency of electron-hole separation, and hence the photocurrent,decreases. For given values of frequency and amplitude of the appliedvoltage, one therefore expects that as the illumination intensityincreases, the current initially increases to a maximum level and thendecreases. Similarly, the impedance initially decreases to a minimumvalue (at maximum current) and then decreases.

This intensity dependence may be used to advantage to induce the lateraldisplacement of beads between fully exposed and partially masked regionsof the interface. As the illumination intensity is increased, the fullyexposed regions will correspond to the regions of interface of lowestimpedance, and hence of highest current, and beads will be drawn intothese regions. As the fully exposed regions reach the state ofdecreasing photocurrent, the effective EIS impedance in those regionsmay exceed that of partially masked regions, with a resulting inversionof the lateral gradient in current. Beads will then be drawn out of thefully exposed regions. Additionally, time-varying changes in theillumination pattern may be used to effect bead motion.

IV—Integration of Biochemical Analysis in a Miniaturized, Planar Format

The implementation of assays in a planar array format, particularly inthe context of biomolecular screening and medical diagnostics, has theadvantage of a high degree of parallelity and automation so as torealize high throughput in complex, multi-step analytical protocols.Minaturization will result in a decrease in pertinent mixing; timesreflecting the small spatial scale, as well as in a reduction ofrequisite sample and reagent volumes as well as power requirements. Theintegration of biochemical analytical techniques into a miniaturizedsystem on the surface of a planar substrate (“chip”) would yieldsubstantial improvements in the performance, and reduction in cost, ofanalytical and diagnostic procedures.

Within the context of DNA manipulation and analysis, initial steps havebeen taken in this direction (i.e. miniaturization) by combining on aglass substrate, the restriction enzyme treatment of DNA and thesubsequent separation of enzyme digests by capillary electrophoresis,see for example, Ramsey, PCT Publication No. WO 96/04547, the contentsof which are incorporated herein by reference, or the amplification ofDNA sequences by application of the polymerase chain reaction (PCR) withsubsequent electrophoretic separation, see, for example, U.S. Pat. Nos.5,498,392 and 5,587,128 to Wilding et at., the contents of which areincorporated herein by reference.

While these standard laboratory processes have been demonstrated in aminiaturized format, they have not been used to form a complete system.A complete system will require additional manipulation such as front-endsample processing, binding and functional assays and the detection ofsmall signals followed by information processing. The true challenge isthat of complete functional integration because it is here that systemarchitecture and design constraints on individual components willmanifest themselves. For example, a fluidic process is required toconcatenate analytical steps that require the spatial separation, andsubsequent transport to new locations, of sets of analyte. Severalpossibilities have been considered including electroosmotic pumping andtransport of droplets by temperature-induced gradients in local surfacetension. While feasible in demonstration experiments, these techniquesplace rather severe requirements on the overall systems lay-out tohandle the very considerable DC voltages required for efficientelectroosmotic mixing or to restrict substrate heating when generatingthermally generated surface tension gradients so as to avoid adverseeffects on protein and other samples.

SUMMARY OF THE INVENTION

The present invention combines three separate functional elements toprovide a method and apparatus facilitating the real-time, interactivespatial manipulation of colloidal particles (“beads”) and molecules atan interface between a light sensitive electrode and an electrolytesolution. The three functional elements are, the electric field-inducedassembly of planar particle arrays at an interface between an insulatingor a conductive electrode and an electrolyte solution; the spatialmodulation of the interfacial impedance by means of UV-mediated oxideregrowth or surface-chemical patterning; and, finally, the real-time,interactive control over the state of the interfacial impedance bylight. The capabilities of the present invention originate in the factthat the spatial distribution of ionic currents, and thus the fluid flowmediating the array assembly, may be adjusted by external intervention.Of particular interest is the introduction of spatial non-uniformitiesin the properties of the pertinent EIS structure. As described herein,such inhomogeneities, either permanent or temporary in nature, may beproduced by taking advantage of the physical and chemical properties ofthe EIS structure.

The invention relates to the realization of a complete, functionallyintegrated system for the implementation of biochemical analysis in aplanar, miniaturized format on the surface of a silicon wafer or similarsubstrate. In addition, the method and apparatus of the presentinvention may be used to create material surfaces exhibiting desirabletopographical relief and chemical functionality, and to fabricatesurface-mounted optical elements such as lens arrays.

The combination of three functional elements endows the presentinvention with a set of operational capabilities to manipulate beads andbead arrays in a planar geometry to allow the implementation ofbiochemical analytical techniques. These fundamental operations apply toaggregates and arrays of colloidal particles including, beaded polymerresins also referred to as Iatices, vesicles, whole chromosomes, cellsand biomolecules including proteins and DNA, as well as metal orsemiconductor colloids and clusters.

Sets of colloidal particles may be captured, and arrays may be formed indesignated areas on the electrode surface (FIGS. 1 a, 1 b and FIGS. 2a-d). Particles, and the arrays they form in response to the appliedfield, may be channeled along conduits of any configuration that areeither embedded in the Si/SiOx interface by U-oxide patterning ordelineated by an external pattern of illumination. This channeling(FIGS. 1 c, 1 d, 1 e, FIGS. 3 c, 3 d), in a direction normal to that ofthe applied electric field, relies on lateral gradients in the impedanceof the EIS structure and hence in the field-induced current. Asdiscussed herein, such gradients may be introduced by appropriatepatterns of illumination, and this provides the means to implement agated version of translocation (FIG. 1 e). The electrokinetic flowmediating the array assembly process may also be exploited for thealignment of elongated particles, such as DNA, near the surface of theelectrode. In addition, the present invention permits the realization ofmethods to sort and separate particles.

Arrays of colloidal particles may be placed in designated areas andconfined there until released or disassembled. The overall shape of thearray may be delineated by UV-oxide patterning or, in real time, byshaping the pattern of illumination. This capability enables thedefinition of functionally distinct compartments, permanent ortemporary, on the electrode surface. Arrays may be subjected to changesof shape imposed in real time, and they may be merged with other arrays(FIG. 1 f) or split into two or more subarrays or clusters (FIG. 1 g,FIGS. 4 a, 4 b). In addition, the local state of order of the array aswell as the lateral particle density may be reversibly adjusted by wayof the external electric field or modified by addition of a second,chemically inert bead component.

The present invention also allows for the combination of fundamentaloperations to develop increasingly complex products and processes.Examples given herein describe the implementation of analyticalprocedures essential to a wide range of problems in materials science,pharmaceutical drug discovery, genomic mapping and sequencingtechnology. Important to the integration of these and otherfunctionalities in a planar geometry is the capability, provided by thepresent invention, to impose temporary or permanentcompartmentalization. In order to spatially isolate concurrent processesor sequential steps in a protocol and the ability to manipulate sets ofparticles in a manner permitting the concatenation of analyticalprocedures that are performed in different designated areas on thesubstrate surfaces.

This invention is for a system and method for programmable illuminationpattern generation. The present invention discloses a novel method andapparatus to generate patterns of illumination and project them ontoplanar surfaces or onto planar interfaces such as the interface formedby an electrolyte-insulator-semiconductor (EIS), e.g., as describedherein. The method and apparatus of the present invention enable thecreation of patterns or sequences of patterns using graphical design ordrawing software on a personal computer and the projection of saidpatterns, or sequences of patterns (“time-varying patterns”), onto theinterface using a liquid crystal display (LCD) panel and an opticaldesign which imagoes the LCD panel onto the surface of interest. The useof the LCD technology in the present invention provides flexibility andcontrol over spatial layout, temporal sequences and intensities (“grayscales”) of illumination patterns. The latter capability permits thecreation of patterns with abruptly changing light intensities orpatterns with gradually changing intensity profiles.

The present invention provides patterns of illumination to control theassembly and the lateral motion of colloidal particles within anenclosed fluid environment. In the presence of a time-varying electricfield applied between two planar electrode surfaces bounding the liquid,particles can be induced to move into or out of illuminated regions ofthe electrode depending on the layout of the patterns, transmitted lightintensity, electric field strength and frequency, junction gapseparation and semiconductor doping levels.

In conjunction with the present invention disclosing a programmableillumination pattern generator, advanced operations of arrayreconfiguration, segmentation and (spatial) encoding are enabled whichin turn lead to a variety of advanced operations and applications.

Applications of the present invention are described in which patternsare generated by projection of fixed masks defining bright and darkareas of illumination of the substrate. The programmable patterngenerator described in the present invention provides flexibility andcontrol over the placement of a plurality of colloidal particles in anovel manner enabling the orchestrated and directed motion of sets ofcolloidal particles. For example, particles assembled into dense planarlayers can be “dragged” and “dropped” interactively by “dragging” and“dropping” the graphical design on a computer screen using a mouse.Alternatively, a sequence of patterns, or a pattern transformation canbe programmed and executed to manipulate arrays of particles in ascheduled manner. Multiple “sub-assemblies” of particles can bemanipulated simultaneously and independently in different areas of thesubstrate under illumination.

BRIEF DESCRIPTION OF DRAWINGS

Other objects, features and advantages of the invention discussed in theabove brief explanation will be more clearly understood when takentogether with the following detailed description of an embodiment whichwill be understood as being illustrative only, and the accompanyingdrawings reflecting aspects of that embodiment, in which:

FIGS. 1 a-h are illustrations of the fundamental operations for beadmanipulation;

FIGS. 2 a and 2 b are photographs illustrating: the process of capturingparticles in designated areas on the substrate surface;

FIGS. 2 c and 2 d are photographs illustrating the process of excludingparticles from designated areas on the substrate surface;

FIGS. 3 a and 3 b are illustrations of the oxide profile of an Si/SiOxelectrode;

FIGS. 3 c and 3 d are photographs of the channeling of particles alongconduits;

FIGS. 4 a and 4 b are photographs of the splitting of an existingaggregate into small clusters;

FIG. 5 is a photograph of the lensing action of individual colloidalbeads;

FIGS. 6 a-c are side view illustrations of a layout-preserving transferprocess from a microtiter plate to a planar cell;

FIG. 7 is a photograph of the inclusion of spacer particles within beadclusters;

FIG. 8 is an illustration of binding assay variations;

FIGS. 9 a and 9 b are illustrations of two mechanisms of particlesorting,

FIG. 10 is an illustration of a planar array of bead-anchoredprobe-target complexes;

FIG. 11 is an illustration of DNA stretching in accordance with thepresent invention;

FIG. 12 is a block diagram of an illumination pattern generatoraccording to the present invention;

FIG. 13 is a block diagram of an illumination pattern generatoraccordance to the present invention;

FIGS. 14 a-d are photographs of different shapes of light inducedarrays;

FIG. 15 a is a photograph of collected particles illustrating particleattraction;

FIG. 15 b is a photograph of confined particles, illustrating particlerepulsion;

FIG. 16 is a photograph illustrating a “drag and drop” operation asapplied to particles;

FIG. 17 is an illustration of the use of an illumination profile tocreate a subarray boundary;

FIGS. 18 a and 18 b are photographs illustrating the setting up andmaintaining of particle confinement patterns;

FIG. 19 is a photograph illustrating the preferential collection of onlyone type of particle present in the mixture into an illuminated areaunder conditions which ensure exclusion of the remainder of theparticles;

FIGS. 20 a-b illustrate the preferential retention of one type ofparticle within an illuminated area under conditions which ensureexpulsion of others using specific combinations of illuminationintensity, frequency and voltage of electric field;

FIGS. 21 a and 21 b are photographs taken at successive times in thecourse of sweeping an illumination pattern across a sample containing aset of small colloidal particles (2.8 μm diameter) which had beendeposited in random positions on a planar substrate surface;

FIGS. 22 a and 22 b are illustrations of methods and procedures ofchemical and spatial encoding of arrays, and methods of decoding arraysby means of selective anchoring of individual beads to substrates,segmentation, and fractionation, respectively;

FIG. 23 is an illustration of random sequential injection;

FIG. 24 is an illustration of sequential injection and light-controlledarray placement;

FIG. 25 a-c illustrate the combined use of chemical and spatial encodingto enhance the encoding complexity of a particle array;

FIG. 26 a-b illustrate a method of producing a composite particle arrayexhibiting a concentric set of discrete bands of composition;

FIG. 27 illustrates the principle of imposing conditions favoringexpulsion of particles from substrate regions illuminated with highintensity;

FIG. 28 illustrates an example with a 4×4 matrix having six fieldspopulated with a random array of beads to produce a unique,miniaturized, non-copyable code; and

FIG. 29 illustrates the light-induced local fluid flow generated at theboundary between illuminated and non-illuminated regions of a substrate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The three functional elements of the present invention may be combinedso as to provide a set of fundamental operations for the interactivespatial manipulation of colloidal particles and molecules, assembledinto planar aggregates adjacent to an electrode surface. In thefollowing description, fundamental operations in this “toolset” aredescribed in order of increasing complexity. Specifically, it is usefulto adopt a classification scheme based on the total number of inputs andoutputs, or “termninals”, involved in a given operation. For example,the merging of two separate arrays, or sets of particles, into one wouldbe a “three-terminal” operation, involving two inputs and one output.The converse three-terminal operation, involving one input and twooutputs, is the splitting of a given array into two subarrays.

Experimental conditions yielding the phenomena depicted in the variousphoto-graphs included herein are as follows. An electrochemical cell isformed by a pair of planar ITO electrodes, composed of an ITO layerdeposited on a glass substrate, or by a Si/SiOx electrode on the bottomand an ITO electrode on the top, separated by a typical gap of 50microns or less. Given its dependence on the photoelectric properties ofthe Si/SiOx interface, light control is predicated on the use of aSi/SiOx electrode. Leads, in the form of platinum wires, are attached tothe ITO and to the silicon electrode, which is first etched to removethe insulating oxide in the contact region, by means of silver epoxy.The cell is first assembled and then filled, relying on capillaryaction, with a suspension of colloidal beads, 1 or 2 microns indiameter, at a typical concentration of 0.1% solids in 0.1 mM azidesolution, corresponding to approximately 2×10^8 particles permilliliter. The number is chosen so as to yield between ½ and 1 fullmonolayer of particles on the electrode surface. Anionic (e.g.,carboxylated polystyrene, silica), cationic (e.g. animated polystyrene)or nominally neutral (e.g. polystyrene) have all been used todemonstrate the basic phenomena underlying the three functional elementsof the present invention. The silicon electrode was fabricated from a 1inch-square portion of a Si (100) wafer, typically 200-250 micronsthick. n-doped to typically 0.01 Ohm) cm resistivity, and capped with athin oxide of typically 30-40 Angstroms thickness. A thick oxide layerof typically 6000-8000 Angstrom thickness, grown under standardconditions in a furnace at 950 degrees C., may be etched by standardphotolithography to define the structures of interest. Alternatively, athin oxide layer may be regrown on a previously stripped surface of(100)-orientation under UV illumination. Given its case ofimplementation to and execution, UV-mediated oxide regrowth is thepreferable technique: it provides the means to pattern the surface byplacing a quartz mask representing the desired pattern in the path of UVillumination and it leaves a chemically homogeneous, topographicallyflat top surface. To avoid non-specific particle adsorption to theelectrode surface, stringent conditions of cleanliness should befollowed, such as those set forth in the General Experimental Conditionsbelow.

The fundamental one-terminal operation is a “capture-and-hold”operation(FIG. 1 a) which forms an array of particles in a designated area ofarbitrary outline on the surface that is delineated by UV-mediated oxidepatterning or by a corresponding pattern of illumination projected on anotherwise uniform Si/SiOx substrate surface. FIGS. 2 a and 2 billustrate bead capture on a surface characterized by a very thin oxideregion 22 (approximately 20-30 Angstroms in thickness) andcorrespondingly low impedance, while the remaining surface is coveredwith the original, thick oxide with correspondingly high impedance. InFIG. 2 a, there is no applied field, and hence, no bead capture. Incontrast, in FIG. 2 b, an electric field is applied (10 Vp-p source, 1kHz) and bead capture occurs within the thin oxide region 22. Underthese conditions, an array starts to grow within less than a second andcontinues to grow over the next approximately 10 seconds as beads arrivefrom increasingly larger distances to add to the outward growingperimeter of region 22. Growth stops when the array approaches the outerlimit of the delineated target area, i.e., the area defined by the thinoxide having a low impedance. The internal state of order of thecaptured aggregate of beads is determined by the strength of the appliedvoltage, higher values favoring increasingly denser packing of bead, andthe eventual formation of ordered arrays displaying a hexagonallycrystalline configuration in the form of a bubble raft. The arrayremains in place as long as the applied voltage is present. Removal ofthe applied voltage results in the disassembly of the array.

The “capture-and-hold” operation may also be implemented underillumination with visible or infrared light for example by simplyprojecting a mask patterned with the desired layout onto the Si/SiOxelectrode. A regular 100 W quartz microscope illuminator has been usedfor this purpose on a Zeiss UEM microscope, with apertures or masksinserted in the intermediate image plane to provide the required shapein the plane of the electrode (when focused properly under conditions ofKoehler illumination). Alternatively an IR laser diode with output of 3mW at 650-680 nm also has been used. The use of external illuminationrather than oxide patterning for the spatial confinement of particlesallows the confinement pattern to be easily modified.

Related to “capture-and-hold” is the one-terminal operation of“exclude-and-hold” (FIG. 1 b) which clears particles from a designatedarea on the surface. Increasing the frequency of the applied voltage toapproximately 100 kHz leads to an inversion in the preference ofparticles which assemble in the thin-oxide portion of the surface (e.g.,region 22, FIG. 2 b) and instead form structures decorating the outsideof the target area perimeter. Rather than relying on this effect, theexclusion of particles from the desired areas is also accomplished, inanalogy to the original “capture-and-hold” operations, by simplyembedding the corresponding structure in the Si/SiOx interface byUV-mediated oxide regrowth. In the example of FIGS. 2 c and 2 d, this isachieved, under conditions otherwise identical to those described above,with respect to FIGS. 2 a and 2 b, by applying 20V (pp) at 10 kHz. Whilethe oxide thickness in the non designated areas 24 is approximately 30Angstroms, the value in the designated square areas 26 is approximately40 Angstroms, implying a correspondingly higher impedance at the appliedfrequency.

The “capture-and-hold” operation enables the spatialcompartmentalization of the substrate surface into functionally distinctregions. For example, particles of distinct chemical type, introducedinto the electrochemical cell at different times or injected indifferent locations, can be kept in spatially isolated locations byutilizing this operation.

The fundamental two-terminal operation is translocation (FIG. 1 c), orthe con-trolled transport of a set of particles from location O tolocation F on the surface, here O and F are target areas to which theabove-described one-terminal operations may be applied. Theone-dimensional, lateral bead transport used in translocation isachieved by imposing a lateral current along a conduit connecting areasO and F, as shown in FIGS. 3 a and 3 b or by projecting a correspondinglinear pattern of illumination. In this channeling operation, beads movein the direction of lower impedance in the direction of the arrow shownin FIGS. 3 a and 3 b, in accordance with the underlying electrokineticflow.

Oxide patterning may be utilized in two ways to create a lateral currentalong the Si/SiOx interface. The simplest method is depicted in FIG. 3 cand shows a large open holding area 32 fed by three narrow conduits 34defined by etching a thermal oxide. Beads move to the holding area 32along the narrow conduits 34 to form a bead array. FIG. 3 d is a largescale view of the array of FIG. 3 c. The principle invoked in creatingtransport is that of changing the aspect ratio (narrow, conduitconnected to wide holding area) of the embedded pattern with constantvalues of thin oxide thickness inside and thick oxide outside, asillustrated in FIG. 3 a. In FIGS. 3 c and 3 d, the applied voltage was10V (pp) at 10 kHz. An alternative approach for creating bead transport,enabled by UV-mediated oxide regrowth, is to vary the oxide thicknessalong the conduit in a controlled fashion. This is readily accomplishedby UV exposure through a graduated filter. Differences in the oxidethickness between O and F of as little as 5-10 Angstroms suffice toeffect lateral transport. In this situation, the aspect ratio of theconduit and holding areas need not be altered. This is illustrated inFIG. 3 b.

The use of external illumination to define conduits, by varying theillumination intensity along the conduit to create the requisiteimpedance gradient, has the advantage that the conduit is only atemporary structure, and that the direction of motion may be modified orreversed if so desired. The present invention provides for mechanisms oflight-mediated active linear transport of planar aggregates of beadsunder interactive control. This is achieved by adjusting an externalpattern of illumination in real time, either by moving the patternacross the substrate surface in such a way as to entrain the illuminatedbead array or by electronically modulating the shape of the pattern toinduce motion of particles.

Two modes of light-mediated, active transport are:

i) Direct Translocation (“tractor beam”) which is a method oftranslocating, arrays and of delineating their overall shape byadjusting parameters so as to favor particle assembly within illuminatedareas of the surface, as described herein. Arrays simply follow theimposed pattern. The rate of motion is limited by the mobility ofparticles in the fluid and thus depends on particle diameter and fluidviscosity.

ii) Transverse Array Constriction is a bead transport mechanism relatedto peristaltic pumping of fluids through flexible tubing. Thelight-control component of the present invention may be used for asimple implementation of this very general concept. A multi-componentplanar aggregate of beads is confined to a rectangular channel, byUV-patterning if so desired, or simply by light. Beads are free to movealone the channel, by diffusion (in either direction). An illuminationpattern matching the transverse channel dimension is set up and is thenvaried in time so as to produce a transverse constriction wave thattravels in one direction along the channel. Such a constriction wave maybe set up in several ways. A conceptually simple method is to project aconstricting mask onto the sample and move the projected mask pattern inthe desired fashion. This method also may be implemented electronicallyby controlling the illumination pattern of a suitable array of lightsources, thus obviating the need for moving parts in the optical train.

The control of lateral bead transport by changing or moving patterns ofillumination has the advantage that it may be applied whenever andwherever (on a given substrate surface) required, without the need toimpose gradients in impedance by predefined UV patterning. On the otherhand, a predefined impedance pattern can provide additional capabilitiesin conjunction with light-control. For example, it may be desirable totransport beads against a substrate-embedded impedance gradient toseparate beads on the basis of mobility.

Conduits connecting O and F need not be straight: as with tracksdirecting the motion of trains. conduits may be shaped in any desirablefashion (FIG. 1 d). A Heated version of translocation (FIG. 1 e) permitsthe transport of particles from O to F only after the conduit is opened(or formed in real time) by a gating signal. This operation utilizes UVoxide patterning to establish two holding areas, O and F, and also lightcontrol to temporarily establish a conduit connecting O and F. Analternative implementation is based on an oxide embedded impedancegradient. A zone along the conduit is illuminated with sufficiently highintensity to keep out particles, thereby blocking the passage, Removal(or reduction in intensity) of the illumination opens the conduit. Inthe former case, light enables the transport of beads, while in thelatter case, light prevents the transport of beads.

The fundamental three terminal operations are the merging and splittingof sets or arrays of beads (FIGS. 1 f and 1 g). The merging of twoarrays (FIG. 1 f) involves the previous two fundamental operations of“capture-and-hold”, applied to two spatially isolated sets of beads inlocations O1 and O2, and their respective channeling; along mergingconduits into a common target area, and their eventual channeling,subsequent to mixing, or a chemical reaction, into the finaldestination, a third holding area, F. This is accomplished, under theconditions stated above, by invoking one-terminal and gated two-terminaloperations.

The splitting of an array into two subarrays (FIG. 1 g) is a specialcase of a generally more complex sorting operation. Sorting involves theclassification of beads in a given set or array into one of two subsets,for example according to their fluorescence intensity. In the simplerspecial case, a given array, held in area O, is to be split into twosubarrays along a demarcation line, and subarrays are to be moved totarget areas F1 and F2. Under the conditions stated above, this isaccomplished by applying the “capture-and-hold” operation to the arrayin O. Conduits connect O to F1 and F2. High intensity illumination alonga narrowly focused line serves to divide the array in a defined fashion,again relying on gated translocation to control transport along conduitsaway from the holding area O. An even simpler version, termedindiscriminate splitting, randomly assigns particles into F1 and F2 bygated translocation of the array in O into F1 and F2 after conduits areopened as described above.

FIGS. 4 a and 4 b show a variant in which beads in region O (FIG. 4 a)are split into multiple regions F1, F2 . . . Fn (FIG. 4 b). Thisreversible splitting of an aggregate or array into n subarrays, orclusters, is accomplished, for carboxylated polystyrene spheres of 2micron diameter at a concentration corresponding to an electrodecoverage of a small fraction of a monolayer, at a frequency of 500 Hz,by raising the applied voltage from typically 5V (pp) to 20V (pp). Thisfragmentation of an array into smaller clusters reflects the effect of afield-induced particle polarization. The splitting is useful todistribute particles in an array over a wider area of substrate forpresentation to possible analytes in solution, and for subsequentscanning of the individual clusters with analytical instruments to makeindividual readings.

The three functional elements of the present invention described hereinmay be also combined to yield additional fundamental operations tocontrol the orientation of anisotropic objects embedded in theelectroosmotic flow created by the applied electric field at theelectrode surface. The direction of the flow, in the plane of thesubstrate, is controlled by gradients in the impedance that are shapedin the manner described in connection with the channeling operation.This is used to controllably align anisotropic objects as illustrated inFIG. 1 b, and may be applied to stretch out and align biomolecules, suchas DNA.

An additional fundamental operation that complements the previous set isthat of permanently anchoring an array to the substrate. This is bestaccomplished by invoking anchoring chemistries analogous to thoserelying on heterobifunctional cross-linking agents invoked to anchorproteins via amide bond formation. Molecular recognition, for examplebetween biotinylated particles and surface-anchored streptavidin,provides another class of coupling chemistries for permanent anchoring.

General Experimental Conditions

The functional elements, namely the electric-field induced assembly ofplanar particle arrays, the spatial modulation of the interfacialimpedance by means of UV-mediated oxide or surface-hemical patterningand finally, the control over the state of the interfacial impedance bylight which are used in the present invention, have been demonstrated inexperimental studies. These studies employed n-doped silicon wafers(resistivities in the range of 0.01 Ohm cm), capped with eitherthermally grown oxide layers of several thousand Angstrom thickness, orwith thin oxide layers, regrown after removal of the original “native”oxide in HF, under UV illumination from a deuterium source in thepresence of oxygen to typical thicknesses between 10 and 50 Angstroms.Lithographic patterning of thermally grown oxide employed standardprocedures implemented on a bench top (rather than a clean room) toproduce features in the range of several microns.

Surfaces were carefully cleaned in adherence with industry standard RCAand Piranha cleaning protocols. Substrates were stored in water producedby a Millipore water purification system prior to use. Surfaces werecharacterized by measuring the contact angle exhibited by a 20microliter droplet of water placed on the surface and viewed (from theside) through a telescope. The contact angle is defined as the anglesubtended by the surface and the tangent to the droplet contour (in sideview) at the point of contact with the surface. For example, a perfectlyhemispherical droplet shape would correspond to a contact angle of 90degrees. Surface chemical derivatization withmercapto-propyl)-trimethoxysilane (2% in dry toluene) produced surfacesgiving typical contact angles of 70 degrees, Oxidation of the terminalthiol functionality under UV irradiation in the presence of oxygenreduced the contact angle to zero in less than 10 nm n of exposure to UVfrom the deuterium source. Other silane reagents were used in a similarmanner to produce hydrophobic surfaces, characterized by contact anglesin excess of 110 degrees.

Simple “sandwich” electrochemical cells were constructed by employingkapton film as a spacer between Si/SiOx and conductive indium tin oxide(ITO), deposited on a thin glass substrate. Contacts to platinum leadswere made with silver epoxy directly to the top of the ITO electrode andto the (oxide-stripped) backside of the Si electrode. In thistwo-electrode configuration, AC fields were produced by a functiongenerator with applied voltages ranging up to 20V and frequenciesvarying from DC to 1 MHZ high frequencies favoring the formation ofparticle chains connecting the electrodes. Currents were monitored witha potentiostat and displayed on an oscilloscope. For convenience,epi-fluorescence as well as reflection differential interferencecontrast microscopy employed laser illumination. Light-inducedmodulations in EIS impedance were also produced with a simple 100 Wmicroscope illuminator as well as with a 3 mW laser diode emitting lightat 650-680 nm.

Colloidal beads, both anionic and cationic as well as nominally neutral,with a diameter in the range from several hundred Angstroms to 20microns, stored in a NaN₂ solution, were employed.

Close attention was paid to colloidal stability to avoid non specificinteractions between particles and between particles and the electrodesurface. Bacterial contamination of colloidal suspensions wasscrupulously avoided.

Typical operating conditions producing, unless otherwise indicated, mostof the results described herein, were: 0.2 mM NaN₂ (sodium azide)solutions, containing particles at a concentration so as to produce notmore than a complete monolayer of particles when deposited on theelectrode; applied DC potentials in the range of 1-4V, and AC potentialsin the range of 1-10V (peak-to-peak) and 500 Hz-10 kHz, with anelectrode gap of 50 microns:anionic (carboxylated polystyrene) beads of2 micron diameter, as well as (nominally neutral) polystyrene beads of2-20 micron diameter.

The method and apparatus of the present invention may be used in severaldifferent areas, examples of which are discussed in detail. Each exampleincludes background information followed by the application of thepresent invention to that particular application.

EXAMPLE I Fabrication of Surfaces and Coatings with Designed Properties

The present invention may be used to fabricate planar surfaces andcoatings with designed properties. Specifically, the functional elementsof the present invention enable the formation of arrays composed ofparticles of a wide range of sizes (approximately 100 Angstrom to 10microns) and chemical composition or surface functionality in responseto AC or DC electric fields. These arrays may be placed and delineatedin designated areas of the substrate, and the interparticle spacing andinternal state of order % within the array may be controlled byadjusting the applied field prior to anchoring the array to thesubstrate. The newly formed surfaces display pre-designed mechanical,optical and chemical characteristics, and they may be subjected tofurther modification by subsequent treatment such as chemicalcross-linking.

The mechanical and/or chemical mortification of surfaces and coatingsprincipally determines the interaction between materials in a wide rangeof applications that depend on low adhesion (e.g., the familiar“non-stick” surfaces important in housewares) or low friction (e.g. toreduce wear in computer hard disks), hydrophobicity (the tendency torepel water, e.g. of certain fabrics), catalytic activity or specificchemical functionality to either suppress molecular interactions withsurfaces or to promote them. The latter area is of particular importanceto the development of reliable and durable biosensors and bioelectronicdevices. Finally, a large number of applications depend on surfaces ofdefined topography and/or chemical functionality to act as templatescontrolling the growth morphology of deposited materials or as “commandsurfaces” directing the alignment of optically active molecules indeposited thin organic films, as in liquid crystal display applications.

Extensive research has been devoted to the formation of surfaces byadsorption of thin organic films of known composition from the liquid orgas phase by several methods. Notwithstanding their seeming simplicityand wide-spread use, these methods can be difficult to handle inproducing reliable and reproducible results. In addition, molecularfilms are not well suited to produce surfaces displaying a regulartopography.

An alternative approach to the problem is the modification of conductivesurfaces by electrophoretic deposition of suspended particulates. Thisis a widely used technique in industrial settings to produce paintcoatings of metal parts, and to deposit phosphor for display screens.The active deposition process significantly enhances the kinetics offormation (in contrast to passive adsorption of organic films fromsolution), an important consideration in practical applications.Electrophoretic deposition requires high DC electric fields and produceslayers in which particles are permanently adsorbed to the surface. Whileparticles in so-deposited monolayers are usually randomly distributed,the formation of polycrystalline monolayers of small (150 Angstrom) goldcolloids on carbon-coated copper grids is also known. However, the useof carbonated copper grids as substrates is not desirable in mostapplications.

Prior art methods have been described for the formation of orderedplanar arrays of particles under certain conditions. For example, theformation of ordered colloidal arrays in response to AC electric fieldson conductive indium tin oxide (TO) electrodes is known. However, theresulting layers were composed of small patches of ordered arrays,randomly distributed over the surface of the otherwise bare ITOsubstrate, Arrays of monodisperse colloidal beads and globular proteinsalso have been previously fabricated by using convective flow andcapillary forces. However, this latter process has the disadvantage ofleaving deposited particle arrays immobilized and exposed to air, makingit difficult to modify arrays by subsequent liquid phase chemistry.

The present invention provides a method of forming planar arrays withprecise control over the mechanical, optical and chemical properties ofthe newly created layer. This method has several distinct advantagesover the prior art. These result from the combination of AC electricfield-induced array formation on insulating electrodes (Si/SiOx) thatare patterned by UV-mediated oxide regrowth. The process of the presentinvention enables the formation of ordered planar arrays from the liquidphase (in which particles are originally suspended) in designatedpositions, and in accordance with a given overall outline. Thiseliminates the above-stated disadvantages of the prior art, i.e., drystate, irregular or no topography, random placement within an aggregate,immobilization of particles and uncontrolled, random placement ofordered patches on the substrate.

An advantage of the present invention is that arrays are maintained bythe applied electric field in a liquid environment. The process leavesthe array in a state that may be readily disassembled, subjected tofurther chemical modification such as cross-linking, or made permanentby chemical anchoring to the substrate. Furthermore, the liquidenvironment is favorable to ensure the proper functioning of manyproteins and protein supramolecular assemblies of which arrays may becomposed. It also facilitates the subsequent liquid-phase deposition ofadditional layers of molecules (by chemical binding to beads or proteinsin the deposited layer), the cycling of arrays between states ofdifferent density and internal order (including complete disassembly ofthe array) in response to electric fields and the chemical cross-linkingof particles into two-dimensionally connected layers, or gels, formed,for example, of chemically functionalized silica spheres. The presentinvention can be practiced on insulating electrodes such as oxide-cappedsilicon, to minimize Faradaic processes that might adversely affectchemical reactions involved in the gelation process or in anchoring, thearray to the substrate. The use of Si/SiOx electrodes also enables thecontrol of array placement by external illumination.

The formation of colloidal arrays composed of small particles inaccordance with the present invention provides a route to thefabrication of surfaces with relief structure on the scale of theparticle diameter. Aside from their optical properties such“micro-rough” surfaces are of interest as substrates for the depositionof DNA in such a way as to alleviate steric constraints and thus tofacilitate enzyme access.

Particles to which the invention applies include silica spheres, polymercolloids, lipid vesicles (and related assemblies) containing membraneproteins such as bacteriorhodopsin (bR)⁻ a light-driven proton pump thatcan be extracted in the form of membrane patches and disks or vesicles.Structured and functionalized surfaces composed of photoactive pigmentsare of interest in the context of providing elements of planar opticaldevices for the development of innovative display and memory technology.Other areas of potential impact of topographically structured andchemically functionalized surfaces are the fabrication of templatesurfaces for the controlled nucleation of deposited layer growth andcommand surfaces for liquid crystal alignment. The present inventionalso enables the fabrication of randomly heterogeneous compositesurfaces. For example, the formation of arrays composed of a mixture ofhydrophobic and hydrophilic beads of the same size creates a surfacewhose wetting and lubrication characteristics may be controlled by thecomposition of the deposited mixed bead array. In this way, the locationof the individual beads is random, but the relative proportion of eachtype of bead within the array is controllable.

EXAMPLE II Assembly of Lens Arrays and Optical Diffraction Elements

The present invention can be used to fabricate lens arrays and othersurface-mounted optical elements such as diffraction gratings. Thefunctional elements of the present invention enable the placement anddelineation of these elements on ITO, facilitating integration withexisting planar display technology, and on Si/SiOx, facilitatingintegration with existing silicon-based device technology.

Silica or other oxide particles, polymer latex beads or other objects ofhigh refractive index suspended in an aqueous solution, will refractlight. Ordered planar arrays of beads also diffract visible light,generating a characteristic diffraction pattern of sharp spots. Thiseffect forms the basis of holographic techniques in optical informationprocessing applications.

A. —The present invention provides for the use of arrays of refractivecolloidal beads as light collection elements in planar array formats inconjunction with low light level detection and CCD imaging. CCD andrelated area detection schemes will benefit from the enhanced lightcollection efficiency in solid-phase fluorescence or luminescencebinding assays.

This assay format relies on the detection of a fluorescence signalindicating the binding of probes to bead-anchored targets in thevicinity of the detector. To maximize through-put, it is desirable tomonitor simultaneously as many binding events as possible. It is herethat array formation by the methods of the present invention isparticularly valuable because it facilitates the placement and Lightpacking of beads in the target area monitored by the CCD detector, whilesimultaneously providing for the additional benefit of lensing actionand the resulting increase in light collection efficiency.

Increased collection efficiency has been demonstrated in experimentsemploying individual, large (10 micron diameter) polystyrene beads aslensing elements to image small (1 micron diameter) fluorescentpolystyrene beads. Under the experimental conditions set forth above anapplied voltage of 5V (pp) at 300 Hz induced the collection of smallparticles under individual large beads within a second. This is shown inFIG. 5, where small beads alone, e.g., 52, appear dim, whereas smallbeads, e.g. 54, gathered under a large bead 56 appear brighter andmagnified. The small beads redisperse when the voltage is turned off.

B. —The use of colloidal bead arrays as diffraction gratings and thus asholographic elements is known. Diffraction gratings have the property ofdiffracting light over a narrow range of wavelengths so that, for givenangle of incidence and wavelength of the illuminating light, the arraywill pass only a specific wavelength (or a narrow band of wave-lengthscentered on the nominal value) that is determined by the inter-particlespacing. Widely discussed applications of diffraction gratings rangefrom simple wavelength filtering to the more demanding realization ofspatial filters and related holographic elements that are essential inoptical information processing.

The present invention provides for a rapid and well controlled processof forming planar arrays in a state of crystalline order which willfunction as surface-mounted optical diffraction elements. In addition,the resulting surfaces may be designed to display topographical reliefto enhance wave-length selective reflectivity. These arrays may beformed in designated areas on a substrate surface. In contrast to theslow and cumbersome prior art method of fabricating such arrays by wayof forming equilibrium crystals in aqueous solutions of low saltcontent, the present invention provides a novel approach to rapidly andreliably fabricate particle arrays at a solid-liquid interface. Thisapproach relies on field-induced formation of arrays to trigger theprocess, and on UV-mediated patterning or light control to position andshape the arrays. In addition, the inter-particle distance, and internalstate of order, and hence the diffraction characteristics of the array,may be fine-tuned by adjusting the applied electric field. For example,a field-induced, reversible order-disorder transition in the array willalter the diffraction pattern from one composed of sharp spots to onecomposed of a diffuse ring. The assembly of such arrays on the surfaceof silicon wafers, as described herein, provides a direct method ofintegration into existing microelectronic designs. Arrays may be lockedin place by chemical coupling to the substrate surface, or by relying onvan der Waals attraction between beads and substrate.

EXAMPLE III A Novel Mechanism for the Realization of a Particle-BasedDisplay

The present invention provides the elements to implement lateralparticle motion as a novel approach to the realization of apanicle-based display. The elements of the present invention provide forthe control of the lateral motion of small particles in the presence ofa pre-formed lens array composed of large, refractive particles.

Colloidal particulates have been previously employed in flat-paneldisplay technology. The operating principle of these designs is based onelectrophoretic motion of pigments in a colored fluid confined betweentwo planar electrodes. In the OFF (dark) state, pigments are suspendedin the fluid, and the color of the fluid defines the appearance of thedisplay in that state. To attain the ON (bright) state, particles areassembled near the front (transparent) electrode under the action of anelectric field. In this latter state, incident light is reflected by thelayer of particles assembled near the electrode, and the display appearsbright. Prototype displays employing small reflective particles inaccordance with this design are known. However, these displays sufferedfrom a number of serious problems including, electrochemical degradationand lack of colloidal stability as a result of prolonged exposure to thehigh DC electric fields required to achieve acceptable switching speeds;and non-uniformities introduced by particle migration in response tofield gradients inherent in the design of the addressing scheme.

The present invention provides a novel mechanism for the design of aparticle-based display which takes advantage of electric field-inducedarray formation as well as controlled, field-induced lateral particledisplacements. First, a lens array composed of colloidal beads isformed. This lens array also serves as a spacer array to maintain awell-defined gap between the bottom electrode and the top electrode thatmay now be placed over the (pre-formed) array. This facilitatesfabrication of uniform flat panel displays with a narrow gap that isdetermined by the particle diameter.

Next, small colloidal particles are added to the electrolyte solution inthe gap. These may be fluorescent, or may be reflecting incident whitelight. Under the action of an AC electric field of appropriatefrequency, these small particles can be moved laterally to assemblepreferentially within the footprint of a larger bead. When viewedthrough a larger bead, small fluorescent beads assembled under a largebead appear bright as a result of the increased light collectionefficiency provided by the lensing action of the large bead; this is theON state (FIG. 5). When moved outside the footprint of the larger bead,particles appear dim, and may be made entirely invisible by appropriatemasking; this is the OFF state. The requisite lateral particle motionmay be induced by a change in the applied voltage or a change in lightintensity. Each large or lensing head introduces a lateral nonuniformityin the current distribution within the electrolyte because the currentis perturbed by the presence of each lensing bead.

In contrast to the prior art displays the present invention employs AC,not DC fields, and insulating (rather than conductive) electrodes,thereby, minimizing, electrochemical degradation. The lateralnon-uniformity introduced by the lens array is desirable because itintroduces lateral gradients in the current distribution within thedisplay cell. These gradients mediate the lateral motion of small beadsover short characteristic distances set by the diameter of the largelensing beads, to effect a switching between ON and OFF states. Thus,the present invention readily accommodates existing technology foractive matrix addressing.

EXAMPLE IV Separation and Sorting of Beads and Particles

The present invention can be used to implement several procedures forthe separation and sorting of colloidal particles and biomolecules in aplanar geometry, Specifically, these include techniques of lateralseparation of beads in mixtures; Individual beads may be removed from anarray formed in response to an electric field by the application ofoptical tweezers.

The separation of components in a given mixture of chemical compounds isa fundamental task of analytical chemistry. Similarly, biochemicalanalysis frequently calls for the separation of biomolecules, beads orcells according to sire and/or surface charge by electrophoretictechniques, while the sorting (most commonly into just two sub-classes)of suspended cells or whole chromosomes according to optical propertiessuch as fluorescence emission is usually performed using field-flowfractionation including flow cytometry and fluorescence-activated cellsorting.

In a planar geometry bead mixtures undergoing diffusion have beenpreviously separated according to mobility by application of an ACelectric field in conjunction with lithographic patterning of theelectrode surface designed to promote directional drift. Essentially, asdescribed in U.S. Pat. No. 5,593,565 to Ajdari et al. the contents ofwhich are included herein by reference, the AC or pulsing electric fieldis used to move small beads in a particular direction over a period oftime, advancing beads of higher mobility relative to those of lowermobility. Capillary electrophoresis has been implemented in a planargeometry, see e.g., B. B. Haab and R. A. Mathies. Anal. Chem 67,3253-33260 (19951) the contents of which are incorporated herein byreference.

The methods of the present invention may be applied in several ways toimplement the task of separation, sorting or isolation in a planargeometry. In contrast to the prior art approaches, the present inventionprovides a significant degree of flexibility in selecting from amongseveral available procedures, the one best suited to the particular taskat hand. In some cases, more than one separation technique may beapplied, and this provides the basis for the implementation oftwo-dimensional separation. That is, beads may be separated according totwo different physical-chemical characteristics. For example, beads mayfirst be separated by size and subsequently, by raising the appliedfrequency to induce chain formation, by polarizability. This flexibilityoffers particular advantages in the context of integrating analyticalfunctionalities in a planar geometry, Several techniques will now bedescribed.

i) The present invention may be used to implement “sieving” in lateral,electric field-induced flow on surfaces patterned by M-UV-mediated oxideregrowth to sort beads in a mixture by size. The fundamental operationsof the invention are invoked to set up directed lateral particle motionalong conduits laid out by UV-mediated oxide regrowth. Conduits aredesigned to contain successively narrower constrictions through whichparticles must pass. Successively finer stages allow only successivelysmaller particles to pass in this “sieving” mechanism (FIG. 9 a). Asshown in FIG. 9 a, the primary particle flow is in the direction left toright, while a transverse flow is established in the top to bottomdirection utilizing an oxide profile as shown. Additionally, rows ofbarriers 92 made from thick oxide are positioned along the conduit withthe spacing between the barriers in each row decreasing in thetransverse direction. As the particles move along the conduit, the rowsof barriers act to separate out smaller particles in the transversedirection. In contrast to previous methods based on electrophoreticseparation, large DC electric fields, and the attendant potentialproblem of electrolysis and interference from electroosmotic flow in adirection opposite to the field-directed particle transport, the presentinvention uses AC electric fields and lateral gradients in interfacialimpedance to produce transport. The present method has the advantage ofavoiding electrolysis and it takes explicit advantage of electroosmoticflow to produce and control particle transport.

In addition, the use of Si/SiOx electrodes enables the use of thelight-control component of the present invention to modify, lateraltransport of beads in real time. For example, external illumination maybe employed to locally neutralize the lateral impedance gradient inducedby UV-mediated oxide regrowth, Particles in these neutral “zones” wouldno longer experience any net force and come to rest. This principle maybe used as a basis for the implementation of a scheme to locallyconcentrate particles into sharp bands and thereby to improve resolutionin subsequent separation.

ii) The present invention may be used to implement “zone refining”, aprocess of excluding minority components of a mixture by size or shapefrom a growing crystalline array of majority component. This processexplicitly depends on the capabilities of the present invention toinduce directional crystallization.

The process of zone re-fining is employed with great success, inproducing large single crystals of silicon of very high purity byexcluding impurities from the host lattice. The concept is familiar fromthe standard chemical procedure of purification by re-crystallization inwhich atoms or molecules that are sufficiently different in size, shapeor charge from the host species so as not to fit into the forming hostcrystal lattice as a substitutional impurity, are ejected into solution.

By enabling the growth of planar arrays, in a given direction and at acontrolled rate, the present invention facilitates the implementation ofan analogous zone refining process for planar arrays. The most basicgeometry is the linear geometry. A multi-component mixture of beads ofdifferent sizes and/or shapes is first captured in a rectangular holdingarea on the surface, laid out by UV-patterning, Next, crystallization isinitiated at one end of the holding area by illumination and allowed toslowly advance across the entire holding area in response to anadvancing pattern of illumination. In general, differences ofapproximately 10% in bead radius trigger ejection.

iii) The present invention may be used to implement fractionation in atransverse flow in a manner that separates particles according tomobility.

Field-flow fractionation refers to an entire class of techniques thatare in wide use for the separation of molecules or suspended particles.The principle is to separate particles subjected to fluid flow in afield acting transverse to the flow. A category of such techniques issubsumed under the heading of electric-field flow fractionation of whichfree-flow electrophoresis is a pertinent example because it iscompatible with a planar geometry. Free-flow electrophoresis employs thecontinuous flow of a replenished buffer between two narrowly spacedplates in the presence of a DC electric field that is applied in theplane of the bounding plates transverse to the direction of fluid flow.As they traverse the electric field charged particles are deflected inproportion to their electrophoretic mobility and collected in separateoutlets for subsequent analysis. In contrast to conventionalelectrophoresis, free-flow electrophoresis is a continuous process withhigh throughput and it requires no supporting medium such as a gel.

The present invention enable; the implementation of field-flowfractionation in a planar geometry. As previously discussed herein,impedance gradients imposed by UV-oxide profiling serve to mediateparticle motion along the electrode surface in response to the externalelectric field. In a cell with a narrow gap, the resultingelectrokinetic flow has a “plug” profile and this has the advantage ofexposing all particles to identical values of the flow velocity field,thereby minimizing band distortions introduced by the parabolic velocityprofile of the laminar flow typically employed in free-flowelectrophoresis.

A second flow field, transverse to the primary flow direction may beemployed to mediate particle separation. This deflecting flow may begenerated in response to a second impedance gradient. A convenientmethod of imposing this second gradient is to take advantage of UV-oxidepatterning to design appropriate flow fields. Both longitudinal andtransverse flow would be recirculating and thus permit continuousoperation even in a closed cell, in contrast to any related prior arttechnique.

Additional flexibility is afforded by invoking the light-controlcomponent of the present invention to illuminate the substrate with astationary pattern whose intensity profile in the direction transverseto the primary fluid flow is designed to induce the desired impedancegradient and hence produce a transverse fluid flow. (FIG. 9 b). This hasthe significant advantage of permitting selective activation of thetransverse flow in response to the detection of a fluorescent beadcrossing a monitoring window upstream. Non-fluorescent beads would notactivate the transverse flow and would not be deflected. This procedurerepresents a planar analog of flow cytometry, or fluorescence-activatedcell sorting.

iv) The invention may be used to induce the formation of particle chainsin the direction normal to the plane of the electrode. The chainsrepresent conduits for current transport between the electrodes andtheir formation may reflect a field-induced polarization. Chains aremuch less mobile in transverse flow than are individual particles sothat this effect may be used to separate particles according to thesurface properties that contribute to the net polarization. The effectof reversible chain formation has been demonstrated under theexperimental conditions stated herein. For example, the reversibleformation of chains occurs for carboxylated polystyrene beads of 1micron diameter, at a voltage of 15 V (pp) at frequencies in excess of 1MHz.

v) The invention may be used to isolate individual beads from a planararray.

Fluorescence binding assays in a planar array format, as describedherein, may produce singular, bright beads within a large array,indicating particularly strong binding. To isolate and retrieve thecorresponding beads, optical tweezers in the form of a sharply focusedlaser spot, may be employed to lock onto an individual bead of interest.The light-control component of the present invention may be used inconjunction with the optical tweezers to retrieve such an individualbead by moving the array relative to the bead, or vice versa, or bydisassembling the array and retaining only the marked bead. This is arather unique capability that will be particularly useful in the contextof isolating beads in certain binding assays.

Commercial instrumentation is available to position optical tweezers inthe field of a microscope. Larger scale motion is facilitated bytranslocating the array in-situ or simply by moving the external samplefixture. This process lends itself to automation in conjunction with theuse of peak-finding image analysis software and feedback control.

vi) The invention may be used to implement a light-induced arraysectioning (“shearing”) operation to separate fluorescent, or otherwisedelineated portions of an array from the remainder. This operation makesit possible to segment a given array and to isolate the correspondingbeads for downstream analysis.

The basis for the implementation of this array segmentation is thelight-control component of the present invention, in the mode of drivingparticles from an area of a Si/SiOx interface that is illuminated withhigh intensity. It is emphasized here that this effect is completelyunrelated to the light-induced force on beads that underlies the actionof optical tweezers. The present effect which operates on large sets ofparticles, was demonstrated under the experimental conditions statedherein using a 100 W illuminator on a Zeiss UEM microscope operated inepi-illumination. A simple implementation is to superimpose, on theuniform illumination pattern applied to the entire array, aline-focussed beam that is positioned by manipulation of beam steeringelements external to the microscope. Beads are driven out of theilluminated linear portion. Other implementations take advantage of twoseparately controlled beams that are partially superimposed. The linearsectioning can be repeated in different relative orientations of shearand array.

EXAMPLE V Fabrication of Spatially Encoded Bead Arrays

The present invention provides a method to transfer suspensions of beadsor biomolecules to the electrode surface in such a way as to preservethe spatial encoding in the original arrangement of reservoirs, mostcommonly the conventional 8×12 arrangement of wells in a microtiterplate. Such a fluid transfer scheme is of significant practicalimportance given that compound libraries are commonly handled andshipped in 8×12 (or equivalent) wells.

The present invention utilizes chemical patterning to define individualcompartments for each of M×N sets of heads and confine them accordingly.In the present instance, patterning is achieved by UV-mediatedphotochemical oxidation of a monolayer of thiol-terminated alkylsilanethat is chemisorbed to the Si/SiOx substrate. Partial oxidation of thiolmoieties produces sulfonate moities and renders the exposed surfacecharged and hydrophilic. The hydrophilic portions of the surface, in theform of a grid of squares or circles, will serve as holding areas.

In accordance with the present invention, the first function ofsurface-chemical patterning into hydrophilic sections surrounded byhydrophobic portions is to ensure trial droplets, dispensed fromdifferent wells, will not fuse once they are in contact with thesubstrate. Consequently, respective bead suspensions will remainspatially isolated and preserve the lay-out of the original M×N wellplate. The second role of the surface chemical patterning of the presentinvention is to impose a surface charge distribution in the form of theM×N grid pattern, which ensures that individual bead arrays will remainconfined to their respective holding areas even as the liquid phasebecomes contiguous.

The layout-preserving transfer procedure involves the steps illustratedin FIGS. 6 a-c. First, as shown in side view in FIG. 6 a, the M×N plateof wells 62 is registered with the pattern 64 on the planar substratesurface. Well bottoms 62, are pierced to allow for the formation ofpendant drops of suspension or, preferably, the process is facilitatedby a fixture (not shown) providing M×N effective funnels to match thegeometric dimensions of the M×N plate on the top and reduce the size ofthe dispensing end. Such a dispensing fixture will also ensure theprecise control of droplet volumes, adjusted so as to slightly overfillthe target holding area on the patterned substrate surface. The set ofM×N drops is then deposited by bringing them in contact with thehydrophilic holding areas of the pre-patterned substrate and relying oncapillary action.

Next, the plate is retracted, and the top electrode is carefully loweredto form the electrochemical cell, first making contact as shown in FIG.6 b, with individual liquid-filled holding areas on the substrate towhich suspensions are confined. Overfilling ensures that contact is madewith individual suspensions. The electric field is now turned on toinduce array formation in the M×N holding areas and to ensure thepreservation of the overall configuration of the M×N sets of beads whilethe gap is closed further (or filled with additional buffer) toeventually fuse individual droplets of suspension into a contiguousliquid phase as shown in FIG. 6 e. In the fully assembled cell of FIG. 1c, while the droplets are fused together, the beads from each dropletare maintained in and isolated in their respective position-s reflectingthe original M×N arrangement of wells. The present invention thusprovides for the operations required in this implementation of alayout-preserving, transfer procedure to load planar electrochemicalcells.

EXAMPLE VI Fabrication of Dynamic Planar Bead Arrays for Parallel Assays

The present invention provides a method to produce a heterogeneous panelof beads and potentially of biomolecules for presentation to analytes inan adjacent liquid. A heterogeneous panel contains particles orbiomolecules which differ in the nature of the chemical or biochemicalbinding sites they offer to analytes in solution. The present methodrelies on the functional elements of the invention to assemble a planararray of a multi-component mixture of beads which carry chemical labelsin the form of tag molecules and may be so identified subsequent toperforming the assay. In the event of binding, the analyte is identifiedby examination of the head, or cluster of beads, scoring positive.

Diagnostic assays are frequently implemented in a planar format of aheterogeneous panel, composed of simple ligands, proteins and otherbiomolecular targets. For example, in a diagnostic test kit, aheterogeneous panel facilitates the rapid testing of a given analyte,added in solution, against an entire set of targets. Heterogeneouspanels of proteins are of great current interest in connection with theemerging field of proteome research. The objective of this research isto identify by scanning the panel with sensitive analytical techniquessuch as mass spectrometry each protein in a multi-component mixtureextracted from a cell and separated by two-dimensional gelelectrophoresis. Ideally, the location of each spot uniquely correspondsto one particular protein. This analysis would permit, for example, thedirect monitoring of gene expression levels in a cell during aparticular point in its cycle or at a given stage during embryonicdevelopment.

The fabrication of an array of heterogeneous targets is central torecently proposed strategies of drug screening and DNA mutation analysisin a planar format. The placement of ligands in a specific configurationon the surface of a planar substrate serves to maintain a key to theidentity of any one in a large set of targets presented simultaneouslyto an analyte in solution for binding or hybridization. In an assayrelying on fluorescence, binding to a specific target will create brightspots on the substrate whose spatial coordinates directly indicate theidentity of the target.

Three principal strategies have been previously employed to fabricateheterogeneous panels. First, protein panels may be created bytwo-dimensional gel electrophoresis, relying on a DC electric field toseparate proteins first by charge and then by size (or molecularweight). Even after many years of refinements this technique yieldsresults of poor reproducibility which are generally attributed to thepoorly defined properties of the gel matrix.

Second, individual droplets, drawn from a set of reservoirs containingsolutions of the different targets, may be dispensed either by hand orby employing one of several methods of automated dispensing (or“printing”; see e.g. Schena et al. Science 270, 467-470 (1995), thecontents of which are incorporated herein by reference). Printing hasbeen applied to create panels of oligonucleotides intended for screeningassays based on hybridization.

Printing leaves a dried sample and may thus not be suitable for proteinsthat would denature under such conditions. In addition, the attendantfluid handling problems inherent in maintaining, and drawing samplesfrom a large number of reservoirs are formidable.

Third, target ligands may be created by invoking a variant of solidphase synthesis based on a combinatorial strategy of photochemicallyactivated elongation reactions. This approach has been limited by veryformidable technical problems in the chemical synthesis of even thesimplest, linear oligomers. The synthesis of non-linear compounds inthis planar geometry is extremely difficult.

The present invention of forming heterogeneous panels requires thechemical attachment of target ligands to beads. Ligands may be coupledto beads “off-line” by a variety of well established coupling reactions.For present purposes, the bead identity must be chemically encoded so itmay be determined as needed. Several methods of encoding, includingbinary encoding, of beads are available. For example) shortoligonucleotides may serve the purpose of identifying a bead via theirsequence which may be determined by microscale sequencing techniques.Alternatively, chemically inert molecular tags may be employed that arereadily identified by standard analytical techniques.

In contrast to all prior art methods, the present invention provides anovel method to create heterogeneous panels by in-situ, reversibleformation of a planar array of chemically encoded beads in solutionadjacent to an electrode. The array may be random with respect tochemical identity but is spatially ordered. This procedure offersseveral advantages. First, it is reversible so that the panel may bedisassembled following the binding assay to discard beads scoringnegative. Positive beads may be subjected to additional analysis withoutthe need for intermediate steps of sample retrieval, purification ortransfer between containers. Second, the panel is formed when needed,that is, either prior to performing the actual binding assay, orsubsequent to performing the assay on the surface of individual beads insuspension. The latter mode minimizes potential adverse effects that canarise when probes bind to planar target surfaces with a highconcentration of target sites. Third to accommodate optical analysis ofindividual beads, interparticle distances within the array may beadjusted by field-induced polarization or by the addition of inertspacer particles that differ in size from the encoded beads. FIG. 7shows the use of small spacer beads 72 for separating encoded beads 74.As shown, the spacing of beads 74 is greater than the spacing ofcomparable beads in FIG. 4 b. Finally, UV-mediated oxide regrowth, asprovided by the present invention, readily facilitates the embedding ofa grid pattern of selected dimension into the substrate to ensure theformation of small, layout-preserving subarrays in the low-impedancefields of the grid.

To create the panel, a multi-component mixture of beads carrying, forexample, compounds produced by bead-based combinatorial chemistry, isplaced between electrodes. Each type of bead may be present in multiplecopies. Arrays are formed in response to an external field in adesignated area of the electrode surface. This novel approach of in-situassembly of panels relies on beads that carry a unique chemical label,or code, to permit their identification subsequent to the completion ofa binding assay. Alternatively, beads may be marked (“painted”) on-lineby way of a photochemical bead-coloring method. Selected beads in anarray are individually illuminated by a focused light source to triggera coloring reaction on the bead surface or in the bead interior toindicate a positive assay score. Beads so marked can be subsequentlyseparated from unmarked heads by a light-activated sorting methoddescribed herein, Numerous UV-activated reactions are available toimplement this bead coloring method.

The present invention provides for several methods of discarding beadswith negative scores, typically the vast majority, while retaining thosewith positive scores. This method take advantage of the fact that, incontrast to all prior art methods, the array represents a temporaryconfiguration of particles that is maintained by the applied electricfield and may be rearranged or disassembled at will. This capabilityalong with the fact that bio-molecules are never exposed to air (as inthe prior art method of printing) facilitates the in-situ concatenationof analytical procedures that require the heterogeneous panel inconjunction with subsequent “downstream” analysis.

First, if positive beads are clustered in a subsection of the array, thelight-controlled array splitting operation of the present invention maybe invoked to dissect the array so as to discard negative portions ofthe array (or recycle them for subsequent use). Second, if positive andnegative beads are randomly interspersed, a fluorescence-activatedsorting method, implemented on the basis of the present invention in aplanar format, as described herein, may be invoked. In the case offluorescence-activated sorting, positive and negative beads may beidentified as bright and dark objects, respectively. In the special casethat only a few positive beads stand out, these may be removed from thearray by locking onto them with optical tweezers, a tool to trap and/ormanipulate individual refractive particles under illumination, anddisassembling the array by removing the field, or subjecting the entirearray to lateral displacement by the fundamental operations of thepresent invention.

The typical task in screening a large set of compounds is one of lookingor a very small number of positive events in a vast number of tests. Theset of discarded beads will typically involve the majority at each stagein the assay. The procedure of the present invention therefore minimizesthe effort invested in negative events, such as the challenging in-situsynthesis of target ligands irrespective of whether or not they willprove to be of interest by binding a probe offered in solution.

The method of forming a heterogeneous panel according to the presentinvention contains beads of each type in generally random assembly. Thecreation of a heterogeneous panel with each position in the panelcontaining a cluster of beads of the same type, that is, beadsoriginating in the same reservoir (FIG. 6 a), may be desirable so as itensure a sufficiently large number of positive events to facilitatedetection. A practical solution follows from the application of thelayout-preserving fluidic transfer scheme described herein. In thisprocedure, beads from an M×N well plate are transferredlayout-preservingly onto a chemically patterned substrate in such a wayas to preserve the spatial encoding of bead identities.

EXAMPLE VII Binding and Functional Assays in Planar Bead Array Format

The present invention can be used to implement mixed-phase bindingassays as well as certain functional assays in a planar array format.Several combinations are possible reflecting the presence of probe ortarget in solution, on the surface of colloidal beads, or on theelectrode surface. The methods of the present invention facilitate theformation of a planar array to present targets to probes in solutionprior to performing the binding assay (“pre formed” array; FIG. 8).Alternatively, a planar array of beads may be formed in front of adetector surface subsequent to performing the binding assay insuspension (“post-formed” array; FIG. 8). The present invention alsoprovides the methods to implement functional assays by enabling theassembly of certain cell types adjacent to a planar detector or sensorsurface to monitor the effects of exposure of the cells to smallmolecule drugs in solution.

Binding assays, particularly those involving proteins such as enzymesand antibodies, represent a principal tool of medical diagnostics. Theyare based on the specific biochemical interaction between a probes suchas a small molecule, and a target, such as a protein. Assays facilitatethe rapid detection of small quantities of an analyte in solution withhigh molecular specificity. Many procedures have been designed toproduce signals to indicate binding, either yielding a qualitativeanswer (binding or no binding) or quantitative results in the form ofbinding or association constants. For example, when an enzyme binds ananalyte, the resulting catalytic reaction may be used to generate asimple color change to indicate binding, or it may be coupled to otherprocesses to produce chemical or electrical signals from which bindingconstants are determined. Monoclonal antibodies, raised from a singlecommon precursor, may be prepared to recognize virtually any giventarget, and immunoassays, based on antibody-antigen recognition andbinding, have developed into an important diagnostic tool. As withenzyme binding, antibody binding of an antigenic analyte may be detectedby a variety of techniques including the classic method of enzyme-linkedimmunoassays (ELISA) in which the reaction of an antibody-coupled enzymeis exploited as an indicator. A common and conceptually simple schemeensures the detection of antibody binding to a target analyte bysupplying a fluorescently labeled second antibody that recognizes thefirst (or primary) antibody.

Binding assays involving soluble globular proteins are often performedin solution to ensure unbiased interactions between protein and target.Such liquid phase assays, especially when performed at lowconcentrations of target or probe, minimize potential difficulties thatmay arise when either target or probe are present in abundance or inclose proximity. By the same token, the kinetics tend to be slow.Cooperative effects, such as crowding, arising from the close proximityof probes must be carefully controlled when either probe or target ischemically anchored to a solid substrate.

Nonetheless, this latter solid phase format of binding assays is alsovery commonly employed whenever the situation demands it. For example,the presence of a protein on the surface of a cell may be exploited in“panning” for the cells that express this protein in the presence ofmany other cells in a culture that do not, desired cells attachthemselves to the surface of a container that is precoated with a layerof a secondary antibody directed against a primary antibody decoratingthe desired cell-surface protein. Similarly, certain phages may begenetically manipulated to display proteins on their surface, and thesemay be identified by a binding assay involving a small molecule probesuch as an antigen if the protein displayed is an antibody (Watson etat., “Recombinant DNA”, 2nd Edition (Scientific American Books, W.H.Freeman and Co., New York N.Y., 1983), the contents of which areincorporated herein by reference). In addition, the planar geometryaccommodates a variety of optical and electrical detection schemesimplemented in transducers and sensors.

A combination of liquid phase and solid phase assay may be developed byusing beads that are decorated with either probe or target, as inprocedures that employ decorated magnetic beads for sample preparationor purification by isolating binding from non-binding molecules in agiven multi-component mixture. Recent examples of the use of these beadsinclude the purification of templates for DNA sequencing applications orthe extraction of mRNAs from (lysed) cells by hybridization to beadsthat are decorated with poly-adenine (polyA) residues.

Functional assays involving suitable types of cells are employed tomonitor extracellular effects of small molecule drugs on cellmetabolism. Cells are placed in the immediate vicinity of a planarsensor to maximize the local concentration of agents released by thecell or to monitor the local pH.

The present invention provides the means to implement mixed phasebinding, assays in a planar geometry with a degree of flexibility andcontrol that is not available by prior art methods. Thus, it offers theflexibility of forming, in-situ, reversibly and under external spatialcontrol, either a planar panel of target sites for binding of analytepresent in an adjacent liquid phase, or a planar array of probe-targetcomplexes subsequent to performing a binding-assay in solution. Bindingmay take place at the surface of individual beads suspended in solution,at the surface of beads pre-assembled into arrays adjacent to theelectrode surface, or at the electrode surface itself, Either the targetor probe molecule must be located on a bead to allow for a bead-basedassay according to the present invention. As shown in FIG. 8, if theprobe molecule P is located on a bead, then the target molecule T may beeither in solution, on a bead or on the electrode surface. The converseis also true.

For example, the methods of the present invention may be used toimplement panning, practiced to clone cell surface receptors, in a farmore expeditious and controlled manner than is possible by the prior artmethod. Given a substrate that has been coated with a layer of antibodydirected against the sought-after cell surface protein, the presentinvention facilitates the rapid assembly of a planar array of cells ordecorated beads in proximity to the layer of antibodies and thesubsequent disassembly of the array to leave behind only those cells orbeads capable of forming a complex with the surface-bound antibody.

A further example of interest in this category pertains to phagedisplays. This technique may be employed to present a layer of proteintargets to bead-anchored probes. Bead arrays may now be employed toidentify a protein of interest. That is, beads are decorated with smallmolecule probes and an array is formed adjacent to the phage display.Binding will result in a probe-target complex that retains beads whileothers are removed when the electric field is turned of, or whenlight-control is applied to remove beads from the phage display. Ifbeads are encoded, many binding tests may be carried out in parallelbecause retained beads may be individually identified subsequent tobinding.

The methods of the present invention readily facilitate competitivebinding assays. For example, subsequent to binding of a fluorescentprobe to a target-decorated bead in solution and the formation of aplanar bead array adjacent to the electrode, fluorescent areas withinthe array indicate the position of positive targets, and these may befurther probed by subjecting them to competitive binding. That is, whilemonitoring the fluorescence of a selected section of the planar array,an inhibitor (for enzyme assays) or other antagonist (of known bindingconstant) is added to the electrochemical cell, and the decrease influorescence originating from the region of interest is measured as afunction of antagonist concentration to determine a binding constant forthe original probe. This is an example of a concatenation of analyticalsteps that is enabled by the methods of the present invention.

The fact that a probe-target complex is fixed to a colloidal bead, as inthe methods of the present invention, conveys practical advantagesbecause this facilitates separation of positive from negative events.Particularly when solid phase assays are performed on a planarsubstrate, an additional advantage of planar bead arrays is theenhancement of light collection efficiency provided by the beads, asdiscussed herein.

If desired, beads may serve strictly as delivery vehicles for smallmolecule probes. That is, an array of probe-decorated beads is formedadjacent to a target-decorated surface in accordance with the methods ofthe present invention. UV-activated cleavage of the probe from the beadsupport will ensure that the probe is released in close proximity to thetarget layer, thereby enhancing speed and efficiency of the assay. Theidentity of the particular probe interacting with the target may beascertained from the positional location of the bead delivering theprobe.

The methods of the present invention apply not only to colloidal beadsof a wide variety (that need no special preparative procedures to makethem magnetic, for example), but also to lipid vesicles and cells thatare decorated with, or contain embedded in their outer wall, eitherprobe or target. The methods of the present invention may therefore beapplied not only to bead-anchored soluble proteins but potentially tointegral membrane receptors or to cell surface receptors.

In particular, the rapid assembly of cells in a designated area of thesubstrate surface facilitates the implementation of highly parallelcell-based functional assays. The present invention makes it possible toexpose cells to small molecule drug candidates in solution and rapidlyassemble them in the vicinity of a sensor embedded in the electrodesurface, or to expose pre-assembled cells to such agents that arereleased into the adjacent liquid phase. In the simplest case, all cellswill be of the same type, and agents will be administered sequentially.Even in this sequential version, electrokinetic mixing will enhancethrough-put. However, as described herein, the methods of the presentinvention also enable the parallel version of binding assays and thus offunctional assays in a planar format by encoding the identity ofdifferent cells by a “Layout-Preserving Transfer” process from an 8×12well plate, as discussed herein, and to isolate cells scoring positiveby providing feed-back from a spatially resolved imaging or sensingprocess to target a specific location in the array of cells.

EXAMPLE VIII Screening for Drug Discovery in Planar Geometry

The functional elements of the present invention may be combined toimplement procedures for handling and screening of compound andcombinatorial libraries in a planar format. The principal requisiteelements of this task arcs sample and reagent delivery from the set oforiginal sample reservoirs, commonly in a format of 8×12 wells in amicrotiter plate, into a planar cell; fabrication of planar arrays oftargets or of probe-target complexes adjacent to the planar electrodesurface prior to or subsequent to performing a binding assay; evaluationof the binding assay by imaging the spatial distribution of markerfluorescence or radioactivity, optionally followed by quantitativepharmacokinetic measurements of affinity or binding constants; isolationof beads scoring positive, and removal from further processing of otherbeads; and collection of specific beads for additional downstreamanalysis. The present invention relates to all of these elements, andthe fundamental operations of the invention provide the means toconcatenate these procedures in a planar format.

A central issue in the implementation of cost-effective strategies formodern therapeutic drug discover) is the design and implementation ofscreening assays in a manner facilitating high throughput whileproviding pharmacokinetic data as a basis to select promising drug leadsfrom a typically vast library of compounds. That is, molecularspecificity for the target, characterized by a binding constant, is animportant factor in the evaluation of a new, compound as a potentialtherapeutic agent. Common targets include enzymes and receptors as wellas nucleic acid ligands displaying characteristic secondary structure.

The emerging paradigm for lead discovery in pharmaceutical and relatedindustries such as agricultural biotechnology, is the assembly of novelsynthetic compound libraries by a broad variety of new methods of solidstate “combinatorial” synthesis. Combinatorial chemistry refers to acategory of strategies for the parallel synthesis and testing ofmultiple compounds or compound mixtures in solution or on solidsupports. For example a combinatorial synthesis of a linear oligopeptidecontaining n amino acids would simultaneously create all compoundsrepresenting the possible sequence permutations of n amino acids. Themost commonly employed implementation of combinatorial synthesis relieson colloidal bead supports to encode reaction steps and thus theidentity of each compound. Beads preferred in current practice tend tobe large (up to 500 microns in diameter) and porous to maximize theircompound storage capacity, and they must be encoded to preserve theidentity of the compound they carry.

Several methods of encoding, or binary encoding, of beads are available.Two examples are as follows. First, beads may be labeled with shortoligonucleotides such as the 17-mers typically employed in hybridizationexperiments. The sequence of such short probes may be determined bymicroscale sequencing techniques such as direct Maxam-Gilbert sequencingor mass spectrometry. This encoding scheme is suitable when the taskcalls for screening of libraries of nucleic acid ligands oroligopeptides. Second, members of a combinatorial library may beassociated with chemically inert molecular tags. In contrast to theprevious case, these tag molecules are not sequentially linked. Instead,the sequence of reaction steps is encoded by the formal assignment of abinary code to individual tag molecules and their mixtures that areattached to the bead in each successive reaction step. The tags arereadily identified by standard analytical techniques such as gaschromatography. This general encoding strategy is currently employed inthe synthesis of combinatorial libraries on colloidal beads.

Commercial compound libraries are large, given that even for theaforementioned 17-mer the number of sequence permutations is 4′17, orapproximately 10′10. However, the high specificity of typical biologicalsubstrate-target interactions implies that the vast majority ofcompounds in the collection will be inactive for any one particulartarget. The task of screening is to select from this large set the fewpotential lead compounds displaying activity in binding or in functionalassays. The principal drug discovery strategy widely applied to naturalcompound libraries in the pharmaceutical industry is to selectindividual compounds from the library at random and subject them to aseries of tests. Systematic screening procedures are thus required toimplement the rapid screening and scoring of an entire library ofsynthetic compounds, in practice often containing on the order of 10′7items.

In current practice, compounds are first cleaved and eluted from theirsolid supports and are stored in microtiter plates. Further samplehandling in the course of screening relies primarily on roboticpipetting and transfer between different containers, typically wells inmicrotiter plates. While robotic workstations represent a step in thedirection of automating the process, they rely on the traditional formatof microtiter plates containing 8×12 wells and sample handling bypipetting, and thus represent merely an incremental operationalimprovement. A significant additional consideration is the need toconserve reagent and sample by reducing the spatial scale of theanalytical procedures.

The present invention provides a set of operations to realize integratedsample handling and screening procedures for bead-based compoundlibraries in a planar format. This will significantly reduce time andcost due to reagent and sample volumes. The principal advantage of themethods of the present invention is that they provide a large set offundamental operations to manipulate sets of beads in a planar format,permitting the handling of beads between stations in a multi-stepanalytical procedure.

In particular, as previously described herein, the methods of thepresent invention facilitate the implementation of the followingpertinent procedures: transfer of samples from microtiter plates to aplanar electrochemical cell; formation of heterogeneous panels of targetsites adjacent to the substrate surface; solid phase binding assays; andisolation of specific beads from an array. In addition, the fundamentaloperations of the present invention provide the means to concatenatethese procedures on the surface of a planar electrode.

As described herein for hybridization assays, several variants arepossible. That is, binding assays may be performed by allowing proteintargets such as enzymes to blind to compounds on the surface of a bead,either in suspension or arranged in a planar array. The common practiceof combinatorial chemistry based on large porous carrier beadsaccommodates the concurrent handling of smaller beads to whose outersurface compounds are anchored via inert chemical spacers. Such smallbeads (up to 10 microns in diameter) are readily manipulated by themethods of the present invention. Large beads are used as labeledcompound storage containers.

Alternatively, binding between target and a radioactively or otherwiselabeled probe may occur in solution, within microtiter plate wells, ifcompounds have already been cleaved from their synthesis support. Inthat case, probe-target complexes may be captured by complexation toencoded beads in each well, for example via the secondary antibodymethod of coupling the protein target to a bead-anchored antibody.Bead-captured probe-target complexes are then transferred to the planarcell for proximity analysis and further processing as illustrated inFIG. 10. As shown in FIG. 10, probe-target complexes 102 are allowed toform in solution. Antibody coated beads 104 are added to the solution,resulting in a bead anchored complex 106. The bead anchored complexes106 are deposited onto electrode 108 from wells 110, and a planar arrayof bead anchored complexes is formed. When fluorescent probes 114 areused, these impart fluorescence to the bead anchored complexfacilitating detection.

The methods and apparatus of the present invention are well suited tothe task of identifying a small number of positive events in a largeset. The imaging of an entire array of probe-target complexes is furtherenhanced by proximity to an area detector, and by bead lensing action.The isolation of a small number of positive scores from the array isreadily achieved, for example by applying optical tweezers, as describedherein. The large remainder of the array may then be discarded. This inturn considerably reduces the complexity of applying more stringenttests, such as the determination of binding constants, because these maybe restricted to the few retained beads. These tests may be directlyapplied, without the need for additional sample transfer to newcontainers, to the samples surviving the first screening pass.

EXAMPLE IX Hybridization Assays in Planar Array Format

The present invention can be used to implement solid phase hybridizationassays in a planar array format in a configuration related to that of aprotein binding assay in which target molecules are chemically attachedto colloidal beads. The methods of the present invention facilitate theformation of a planar array of different target oligonucleotides forpresentation to a mixture of strands in solution. Alternatively, thearray may be formed subsequent to hybridization in solution tofacilitate detection and analysis of the spatial distribution offluorescence or radioactivity in the array.

Considerable research and development is presently being invested in aneffort to develop miniaturized instrumentation for DNA sample extractionand preparation including amplification, transcription, labeling andfragmentation, with subsequent analysis based on hybridization assays aswell as electrophoretic separation. Hybridization assays in planar arrayformat are being developed as a diagnostic tool for the rapid detectionof specific single base pair mutations in a known segment of DNA, andfor the determination of expression levels of cellular genes viaanalysis of the levels of corresponding, mRNAs or cDNAs. Hybridizationof two complementary single strands of DNA involves molecularrecognition and subsequent hydrogen bond formation between correspondingnucleobases in the two opposing strands according to the rules A-T andG-C; here A, T, G and C respectively represent the four nucleobasesAdenine, Thymine, Guanosine and Cytosine found in DNA; in RNA, Thymineis replaced by Uracil. The formation of double-strand, or duplex, DNArequires the pairing of two highly negatively charged strands of DNA,and the ionic strength of the buffer, along with temperature, plays adecisive role.

As previously discussed herein, two principal methods to prepareheterogeneous arrays of target strands on the surface of a planarsubstrate are micro-dispensing (“printing”) and in-situ, spatiallyencoded synthesis of oligonucleotides representing all possible sequencepermutations for a given total length of strand, in this context,hybridization must necessarily occur in close proximity to a planarsubstrate surface and this condition requires care if complications fromsteric hindrance and from non-specific binding of strands to thesubstrate are to be avoided. Non-specific adsorption can be a seriousproblem especially in the presence of DC electric fields employed incurrent commercial designs that rely on electrophoretic deposition toaccelerate the kinetics of hybridization on the surface. In addition,there are the technical difficulties, previously discussed herein,resulting from steric hindrance and from collective effects reflectingthe crowding of probe strands near the surface.

In the context of DNA analysis, colloidal (magnetic) beads are commonlyused. For example, they are employed to capture DNA in a widely usedscreening procedure to select cDNAs from clone libraries. Specifically,cDNAs are allowed to hybridize to sequences within long genomic DNA thatis subsequently anchored to magnetic beads to extract the hybridizedcDNA from the mixture.

The present invention facilitates the formation of planar arrays ofoligonucleotide-decorated colloidal beads, either prior to or subsequentto hybridization of a fluorescence probe strand to the bead-anchoredtarget strand or subsequent to hybridization in free solution and beadcapture of the end-functionalized target strand. In contrast to priorart methods, the present invention does not require hybridization tooccur in the vicinity of planar substrate surface, although this is anoption if bead-anchored probe strands are to be delivered tosubstrate-anchored target strands.

The ability to perform hybridization either in solution, on the surfaceof individual beads, or at the substrate surface provides anunprecedented degree of flexibility. In addition, the advantages of beadarrays, as described herein, make it feasible to select and isolateindividual beads, or groups of beads, from a larger array on the basisof the score in a hybridization assay. This isolation facilitates theimplementation of subsequent assays on the strands of interest. The factthat beads remain mobile also means that beads of interest may becollected in designated holding areas for micro-sequencing, or may bemoved to an area of substrate designated for PCR amplification.

The methods of the present invention may be used to implement ahybridization assay in a planar array format in one of two principalvariations. All involve the presence of the entire repertoire of beadsin the planar array or panel formed adjacent to the electrode surfacefor parallel read-out. As with heterogeneous panels in general, thearrangement of beads within the array is either random (with respect tochemical identity), and the identity of beads scoring high in thebinding assay must be determined subsequently or it is spatially encodedby invoking the “Layout-Preserving Transfer” method of sample loadingdescribed herein.

The former variant is readily implemented and accommodates arrayformation either prior to or subsequent to performing the binding assay.For example, binding may be performed in suspension before beads areassembled into the array. As with the aforementioned cDNA selectionprocedure, the method of the preset invention also accommodates the useof beads as capture elements for end-functionalized target DNA, forexample, via biotin-streptavidin complexation. In this latter case,beads serve as a delivery vehicle to collect all probe-target complexesto the electrode surface where they are assembled into an array for easeof analysis. In particular, proximity CCD detection of beads onelectrodes will benefit from the lensing action of the beads in thearray. This version of the assay is preferably used if only a smallnumber of positive scores are expected.

Hybridization to a pre-formed bead array can take advantage of a variantof the assay which preserves spatial encoding. An array of bead clustersis formed by the “Layout-Preserving Transfer” method previouslydescribed herein, and exposed to a mixture of cDNAs. The resultingspatial distribution of fluorescence intensity or radioactivity reflectsthe relative abundance of cDNAs in the mixture. This procedure relies onthe detection of a characteristic fluorescence or other signal from theprobe-target complex on the surface of a single bead.

Given the fact that the array is readily held stationary by the methodsof the present invention, image acquisition may be extended to attainrobust signal-to-noise for detection of low level signals. For example,a signal generated by a bead of 10 micron diameter with at most 10^8probe-target complexes on the surface of the bead may be detected. Beadlensing action also aids in detection.

As with the implementation of drug screening, the functional elements ofthe present invention may be combined to perform multiple preparativeand analytical procedures on DNA.

EXAMPLE X Alignment and Stretching of DNA in Electric Field-Induced Flow

The present invention can be used to position high-molecular weight DNAin its coiled configuration by invoking the fundamental operations asthey apply to other colloidal particles. However in addition, theelectrokinetic flow induced by an electric field at a patternedelectrode surface may be employed to stretch out the DNA into a linearconfiguration in the direction of the flow.

Procedures have been recently introduced which rely on optical imagingto construct a map of cleavage sites for restriction enzymes along thecontour of an elongated DNA molecule. This is generally known as a“restriction map”. These procedures, which facilitate the study of theinteraction of these and other proteins with DNA and may also lead tothe development of techniques of DNA sequencing, depend on the abilityto stretch and align DNA on a planar substrate.

For individual DNA molecules, this has been previously achieved bysubjecting the molecule to elongational forces such as those exerted byfluid flow magnetic fields acting on DNA-anchored magnetic beads orcapillary forces. For example, DNA “combs” have been produced by simplyplacing, DNA molecules into an evaporating droplet of electrolyte. Ifprovisions are made to promote the chemical attachment of one end of themolecule to the surface, the DNA chain is stretched out as the recedingline of contact between the shrinking droplet and the surface passesover the tethered molecules. This leaves behind dry DNA molecules thatare attached in random positions within the substrate area initiallycovered by the droplet, stretched out to varying degrees and generallyaligned in a pattern of radial symmetry reflecting the droplet shape.Linear “brushes”, composed of a set of DNA molecules chemically tetheredby one end to a common line of anchoring points, have also beenpreviously made by aligning and stretching DNA molecules bydielectrophoresis in AC electric fields applied between two metalelectrodes previously evaporated onto the substrate.

The present invention invokes electrokinetic flow adjacent to anelectrode patterned by UV-mediated regrowth of oxide to provide a novelapproach to the placement of DNA molecules in a predeterminedarrangement on a planar electrode surface, and to the stretching of themolecules from their native coil configuration into a stretched, linearconfiguration that is aligned in a pre-determined direction. Thisprocess is shown in FIG. 11 and is accomplished by creating controlledgradients in the flow vicinity across the dimension of the DNA coil. Thevelocity gradient causes different portions of the coil to move atdifferent velocities thereby stretching out the coil. By maintaining astagnation point at zero velocity, the stretched coil will be fixed inposition. This method has several advantages over the prior artapproaches. First DNA molecules in their coiled state are subjected to)light control to form arrays of desired shape in any position on thesurface. This is possible because large DNA from cosmids or YACs formscoils with a radius in the range of one micron, and thus acts in amanner analogous to colloidal beads. A set of DNA t-molecules may thusbe steered into a desired initial arrangement. Second, UV-patterningensures that the elongational force created by the electrokinetic flowis directed in a predetermined direction. The presence of metalelectrodes in contact with the sample, a disadvantage of thedielectrophoretic prior art meCthod, is avoided by eliminating thissource of contamination that is difficult to control especially in thepresence of an electric field. On patterned Si/SiOx electrodes, flowvelocities in the range of several microns/second have been generated,as required for the elongation of single DNA molecules it flow. Thus,gradients in the flow field determines both the fractional elongationand the orientation of the emerging linear configuration. Third, thepresent invention facilitates direct, real-time control of the velocityof the electric field-induced flow, and this in turn conveys explicitcontrol over the fractional elongation.

This invention is for a system and method for programmable illuminationpattern generation. The present invention discloses a novel method andapparatus to generate patterns of illumination and project them ontoplanar surfaces or onto planar interfaces such as the interface formedby an electrolyte-insulator-semiconductor (EIS), e.g., as describedherein. The method and apparatus of the present invention enable thecreation of patterns or sequences of patterns using graphical design ordrawing software on a personal computer and the projection of saidpatterns, or sequences of patterns (“time-varying patterns”), onto theinterface using a liquid crystal display (LCD) panel and an opticaldesign which images the LCD panel onto the surface of interest. The useof the LCD technology in the present invention provides flexibility andcontrol over spatial layout, temporal sequences and intensities (“grayscales”) of illumination patterns. The latter capability permits thecreation of patterns with abruptly changing light intensities orpatterns with gradually changing intensity profiles.

The present invention provides patterns of illumination to control theassembly and the lateral motion of colloidal particles within anenclosed fluid environment. In the presence of a time-varying electricfield applied between two planar electrode surfaces hounding the liquid,particles can be induced to move into or out of illuminated regions ofthe electrode depending on the layout of the patterns, transmitted lightintensity, electric field strength and frequency, junction gapseparation and semiconductor doping levels.

In conjunction with the present invention disclosing a programmableillumination pattern generator, advanced operations of arrayreconfiguration, segmentation and (spatial) encoding are enabled whichin turn lead to a variety of advanced operations and applications.

Applications of the present invention are described in which patternsare generated by projection of fixed masks defining bright and darkareas of illumination of the substrate. The programmable patterngenerator described in the present invention provides flexibility andcontrol over the placement of a plurality of colloidal particles in anovel manner enabling the orchestrated and directed motion of sets ofcolloidal particles. For example, particles assembled into dense planarlayers can be “dragged” and “dropped” interactively by “dragging” and“dropping” the graphical design on a computer screen using a mouse.Alternatively, a sequence of patterns, or a pattern transformation canbe programmed and executed to manipulate arrays of particles in ascheduled manner. Multiple “sub-assemblies” of particles can bemanipulated simultaneously and independently in different areas of thesubstrate under illumination.

The programmable illumination pattern generator according to the presentinvention includes a liquid crystal display (LCD) panel serving as aspatially addressable mask which perm its multiple levels oftransmission for each of an array of individually addressable pixels viainterface control and drive electronics receiving an output generated byvideo graphics adapters, such as those commonly used with personalcomputers. The LCD panel contains an array of pixels which areindividually programmed to transmit a portion of light intensityincident upon the pixel. Available LCD technology permits the control oftransmissivity in 256 levels (“gray scales”) and the change of theentire pattern, composed of 240×320 pixels arranged in a 4 mm by 6 mmpanel under active matrix addressing. Such displays include, forexample, Cyberbisplay KCD-QK01 AA. 320 Evaluation Kit, available fromKopin Corp. Tatunton. MA. The LCD panel drive electronics receives inputfrom the PC in the form of VGA or other graphics output that drives thesystem monitor.

An optical design and instrumental implementation of a combined opticalprojection and imaging apparatus projecting a programmed configurationof the LCD panel (“mask”) into the field of view of an optical imaginginstrument which is capable of microscopic image construction by way ofmultiple contrast mechanisms is shown in FIGS. 12 and 13. FIG. 12 is ablock diagram illustrating the layout of a programmable illuminationpattern generator combining projection and imaging optics, LCDprojection display technology with a software control and applicationsuite to create spatially and temporally con-trolled illuminationpatterns and produce a demanified projected image of these patterns inthe field of view of an imaging system utilizing an observation camera.FIG. 13 is a block diagram illustrating a programmable illuminationpattern generator halving illumination and on-line inspectionsubsystems. The illumination train contains a light source, such as alaser diode or other collimated light source, and is configured inaccordance with standard Koehler illumination so as to image the LCDpanel into the object plane of the objective lens. The on lineinspection (“imaging”) system invokes bright-field, dark-field orfluorescence contrast to produce an image of sample and superimposedprojected LCD pattern on the face of a CCD (or other) imaging device.Illuminating and imaging rays are shown in FIG. 13.

The apparatus according to the present invention may be implementedusing National Instruments' LabView (Vs. 5.1) software which provides agraphical user interface Application software modules developed withLabView enable the construction and projection of:

-   -   “still” frames (static spatial control of particles) loaded from        a graphics file or created interactively    -   a sequence of frames (dynamic spatial and temporal control),        each composed of a grayscale image, and applied to an assembly        of particles via the projection system by way of a at least one        of:        -   a “drag-and drop” operation applied with a “mouse” to a            single graphics feature (“shape”);        -   creating, storing and playing back a “trajectory” for a            shape;        -   loading a sequence of procreated image files

Specific adaptations of this general purpose design are possible incertain applications and include, the use of:

-   -   static illumination sources such as laser diodes arranged in a        pre-determined configuration    -   a scanning spot or line when repetitive, long-range “drag-and        drop” operations are to be performed

The apparatus of the present invention provides a set of advancedoperations for single arrays or for a multiplicity of disjoint arraysmaintained within a common fluid phase (“subarrays”). These operationsinclude reconfiguration segmentation and (spatial) encoding,(“subarrays”), which are described in detail below.

In the case of reconfiguration, arrays of particles may be reconfiguredin-situ by adjusting the shape and outlines of projected patterns ofillumination, as illustrated in FIGS. 14 a-d. FIGS. 14 a-d illustrateexamples of light-induced adjustments, in the overall shape of arrayscomposed of assembled 2.2 μm-diameter colloidal particles, imaged hereusing dark field contrast. Arrays such as those shown here are formed inresponse to a combination of an AC field (typically 1-5 V (peak-to-peak)and 0.1-10 kHz) and illumination delivered to a silicon substrate ofintermediate doping level (typically in the range of 0.01 to 5 Ohm cm)and coated with a thin (<100 Å) oxide; aqueous media such as water orweak electrolyte solutions (typically containing less than 1 OMNI salt)or non-aqueous media such as DMSO may be used. The sequence of shapetransformations was produced by first assembling particle within acircular illuminated area (FIG. 14 a) and then successively changing theshape of the projected illuminated area using application softwaredescribed herein. Specifically, a vertical rectangle (FIG. 14 b),horizontal rectangle (FIG. 14 c) and a square (FIG. 14 d). Under thecited conditions, particles in the range of 2.2 μm diameter such thoseshown here respond to the imposed changes within at most a few seconds.

The programmable methods according to the present invention facilitatethe implementation of complex array reconfigurations. Specifically, an“attraction” mode (FIG. 15A) and a “rejection” mode (FIG. 15B) may beachieved, wherein the intensity of illumination is adjusted, inconjunction with the selection of suitable frequencies of the appliedelectric field, to either induce particles to move and remain stationarywithin illuminated areas (FIG. 15A) or to move out of illuminated areas(FIG. 15B). Multiple fundamental shapes can be combined into complexshapes to construct regions in which particles are confined (“trapped”)(FIG. 15B). These confinement areas serve as local reservoirs from whicha desired number of particles can be released under light control.

FIGS. 15 a-b illustrate the capabilities over control of particleposition and array assembly and reconfiguration according to the presentinvention, namely, collection and array assembly within illuminatedsubstrate regions and expulsion of panicles from illuminated substrateregions to discrete locations delineating the shape of the illuminatedregion. In this example, 2.2 μm particles, were imaged using dark-fieldcontrast, and assembled within a region shaped in the form of arectangular frame as well as within a circular region contained withinthe frame. Operating conditions were: 1 kHz/10V p-p. When the incidentillumination intensity is increased by 20% under otherwise unchangedconditions, particles are expelled from both illuminated regions andinstead collect in a region surrounding the frame shape (on eitherside). Particles expelled to the interior portion of the frame andparticles expelled from the central circular shape are confined in theintervening space where they assemble into an array whose inner andouter contours respectively trace the circular interior shape and therectangular exterior shape of the most proximal illuminated regions.

Expulsion can be induced by increasing the illumination intensity atconstant frequency, ω, as long as ω<ω_c a characteristic frequency.Alternatively, expulsion can be induced by increasing the frequency atconstant illumination intensity to a value ω<ω_c (see also FIGS. 20 a-bbelow). The frequency ω_c is a characteristic dielectric relaxationfrequency associated with the field-induced particle polarization whichis in turn determined by interfacial polarization of the particle andreflects physical-chemical properties of each particle primarilyincluding its weight shape size and electric susceptibility (relative tothat of the suspending medium), a property which in turn reflects theparticle's surface-chemical composition. The width of the visible (dark)bands depleted of particles is determined by a combination of V p-p,frequency, applied DC bias voltage and illumination intensity. Theexpulsion mechanism enables precision control over particle number andposition.

As with fundamental shapes, complex shapes can be “dragged” and“dropped” to transport confined assemblies of particles to desiredpositions on the substrate (FIG. 16). The method and apparatus disclosedherein permit several implementations to “drag-and-drop” particleassemblies. These include: interactively moving a mouse/cursor;laterally displacing the sample relative to a stationary illuminationpattern; or laying out intensity profiles to direct the lateraltransport of an assembly of particles to a final destination on thesubstrate. To favor either a close-packed array configuration or anexpanded assembly configuration of a set of particles, light intensityas well as voltage and frequency of the alternation electric field maybe adjusted. An expanded configuration is favored for the “drag”operation, a close-packed configuration provides stability following the“drop” operation.

FIG. 16 illustrates three consecutive steps of merging, within acontiguous fluid suspending medium, a set of initially three packets ofbeads (top row), into two packets by merging the left and center packets(middle row) into finally a single packet by merging the left with thecentral packets (bottom row). This illustrates the power of “drag-anddrop” as well as merge operations. Particles and operating conditions inthis example are similar to those in FIGS. 15 a-b.

In the case of segmentation, operations such as those described above,enable procedures to fractionate mixtures of particles on the basis ofshape, size and electrochemical properties such as surface charge andpolarizability and to segment arrays into subarrays in order to isolateand retrieve specific particles of interest. FIG. 17 illustrates aparticular sequence of operations to isolate a patch off fluorescentparticles from a previously formed array. This segmentation operation isimplemented by applying a sequence of illumination patterns projectinghigh intensities into positions from which particles are to be excluded,thereby segmenting an array into subarrays according to the projectedpattern

For example, an array may be sectioned into subarrays by applying asequence of high-intensity “lines” each acting as a “scalpel” (FIG. 17).By iterating this operation small sets of particle(s) of interest may beisolated and retrieved from an array by illuminating a region containingthese particle(s), then successively subdividing the region (FIG. 17).FIG. 17 illustrates the process of segmenting an array by projecting anillumination pattern of high intensity in the shape of a vertical line,thereby excising a highlighted regions of interest within the array.Individual steps are shown from left to right, with the iterationleading to complete excision.

Also, multiple individual particles may be maintained and manipulatedwithin the field by setting tip and maintaining confinement patterns(FIGS. 18 a and 18 b). The resulting capability is analogous to that ofa multi-point “optical tweezer”, In fact. “optical tweezers” may beapplied in conjunction with the method and apparatus disclosed herein tolock onto specific individual particles using a focused laser beam andgalvanometric mirror.

As shown in FIGS. 18 a-b, control over individual particles and cellsmay be achieved by providing optical confinement under conditionsensuring collection of particles into illuminated regions (see FIG. 15a). When two illuminated regions are brought, into proximity using“drag-and drop”, individual particles can be transferred betweenadjacent confinement regions (“traps”). The direction of transfer isdetermined by small differences in illumination intensity: the brighterregion is preferred (under conditions ensuring that the regime ofexpulsion (FIG. 15 b) is avoided. In the example, particles areexchanged as shown between two illuminated confinement regions: in theinitial state (FIG. 18 a), one particle is confined in the verticalilluminated rectangle, three in the horizontal illuminated rectangle, inthe Final state (FIG. 18 b), this configuration has been inverted.Particles and operating conditions in this example are similar to thosein FIGS. 18 a-b.

The fractionation of a heterogeneous mixture of particles composed ofmultiple types of particles may be accomplished by creating adifferential response of different particle types to the various forcesacting on them. Physical-chemical particle properties of interestinclude size, shape and electric polarizability. Operating parametersinclude illumination intensity, frequency and voltage of the alternatingelectric field, as well as silicon substrate doping levels.

Referring now to FIG. 19, therein is illustrated the preferentialcollection of only one type of particle present in the mixture into anilluminated area under conditions which ensure exclusion of theremainder of the particles. FIG. 19 illustrates fractionation of amixture of particles by preferential collection of one of two particletypes into a circular illuminated region. Under suitable conditions (seediscussion of expulsion and characteristic frequencies in connectionwith FIGS. 20 a-b below), particles of 3.2 μm diameter are collectedinto the illuminated region and assemble into an array, while particlesof 4.5 μm diameter are expelled from this region, assembling, intostrings pointing radially outward from the central region and lining theperimeter of the region. Operating conditions in this example aresimilar to those used in connection with FIGS. 20 a-b.

Similarly, FIG. 20 a illustrates the preferential retention of one typeof particle within an illuminated area under conditions which ensureexpulsion of others using, specific combinations of illuminationintensity, frequency and voltage of electric field. Particles ofdiffering size or electric polarizability exhibit characteristicfrequencies such that when the frequency of the applied electric fieldis lowered to this characteristic value, the corresponding type ofparticle is expelled.

FIG. 20 a is composed of four sub-panels and illustrate the concept offractionating a heterogeneous mixture of particles into its constituenthomogeneous particle subpopulations by invoking the differentialfrequency dependence of particle expulsion from illuminated substrateregions. For a single particle type, FIG. 20 a, top left and bottom leftillustrate that particles are collected into an illuminated region whento, the frequency of the applied electric field, is set to a value belowω_c, a characteristic relaxation frequency, while particles are expelledwhen ω>ω_c (see also FIGS. 15 a-b). For a mixture of two particle types,(FIG. 20 a, top right and bottom right)f with the types differing in oneof their physical-chemical properties, including size and electricsusceptibility and correspondingly differing in their characteristicfrequencies ω_c(type 1)<ω_c(type 2), a novel process of fractionation isillustrated. Specifically, the expulsion of a single type of particlefrom the illuminated region is induced under conditions ensuring thatthe second type remains confined.

FIG. 20 b illustrates an actual realization of fractionation analogousto that depicted in the bottom right subpanel of FIG. 20 a using twotypes of beads. 3.2 μm and 4.5 μm in diameter respectively. In thisexample, the actual realization proceeds from an initial state in whichparticles of both types are placed randomly on the substrate surface. Acircular region in the center of the field was illuminated underconditions of intensity, AC voltage (approximately n V p-p) andfrequency (approximately 1 kHz) so as to induce the assembly of an arraycomposed exclusively of the smaller particles within the illuminatedregion and simultaneously to induce expulsion of the larger particles ina radially outward direction. As a result, expelled particles aretrapped in a diffuse “ring” of recirculating fluid flow.

An additional capability is that of sweeping an illumination pattern(“shape”) across the field of view under conditions enablingpreferential collection of a single type of particle into theilluminated area, thereby physically separating the designated type ofparticle from a given random mixture and enriching and depositing saiddesignated particle type in a target location. This is illustrated inFIGS. 21 a-b which illustrate snapshots taken at successive times in thecourse of sweeping an illumination pattern across a sample containing aset of small colloidal particles (2.8 μm diameter) which had beendeposited in random positions on a planar substrate surface. As thepattern moves from the left (FIG. 21 a) to the right (FIG. 21 b),particles collect within the illuminated region of the surface (FIG. 21a). Typical operating conditions include an applied peak-to-peak voltageof 1-10V, typical frequencies of 0.1-5 kHz (depending on the size andsurface-chemical properties of the particles of interest) and lightintensities delivered by a 100 mW laser diode emitting at 670 nm. In theexample shown in FIGS. 21 a-b, the projected pattern was swept acrossthe field of view of 400 μm in approximately 20 s. As the illuminationpattern is moved, particles track this movement while additionalparticles are swept up in the pattern, leaving a swept region frontwhich particles have been substantially removed. Differences inphysical-chemical particle properties including mass, size, surfacemorphology or electrochemical properties including electricpolarizability can lead to differential particle mobility. In theexample shown in FIGS. 21 a-b, trailing particles spread out behind themoving illumination pattern (FIG. 21 b). A lower particle mobility leadsto a wider tail as slower particles fall farther behind. Thisdifferential particle mobility can be used to fractionate aheterogeneous mixture of particles into constituent particle populationsA similar fractionation capability is attained by invoking illuminationprofiles such that the illumination intensity exhibits a prescribedspatial variation.

A particularly versatile method of fractionating a heterogeneous randommixture of beads into multiple constituent populations is the creationof illumination intensity gradients, wherein frequencies of appliedelectric field are selected so as to allow multiple types of particlesto come to rest in distinct and characteristic locations within theintensity gradient.

While segmentation primarily relates to “post-processing” arraysfollowing an assay, “pre-processing”, of arrays ensures a uniqueencoding of a plurality of chemical identities of molecules displayed onthe surfaces of beads within the array. FIGS. 22 a-b provide an overviewof this process. In contrast to conventional methods such as “printing”or “spotting” of arrays of antibodies or DNA and by in-situ chemicalsynthesis of oligonucleotides the present invention discloses methodsand apparatus to produce spatially and chemically encoded planar arraysof particles in which chemical compounds (including but not limited toproteins including antibodies or antigens, oligonucleotides, DNA andRNA) are displayed on bead surfaces and are NOT attached to thesubstrate on which the array is assembled.

FIGS. 22 a-b provide an overview of methods and procedures of chemicaland spatial encoding of arrays (FIG. 22 a) and methods of decodingarrays by means of selective anchoring of individual beads tosubstrates, segmentation, and fractionation (FIG. 22 b) to enable theunique in-situ determination of the chemical identity of each of thebeads within an assembled array. The former methods and procedures areof particular interest in “pre-processing” of arrays prior to their usein test procedures (“assays”) or in concurrent processing; the latterare of particular interest in “post-processing” of arrays following anassay. Methods of spatial encoding are elaborated below (see also FIGS.23 and 24 for details on Sequential Injections). Methods of decoding, or“post-processing” of arrays follow by way of segmentation andfractionation, as discussed with respect to FIGS. 17, 19 and 20 a-b.

According to the present invention, chemical and spatial encoding may becombined to encode and decode the identities (“types” of particles suchas colloidal beads within a planar array. That is, discrete “packets” ofbeads, originating in a common reservoir and containing a plurality ofchemically encoded bead types, are maintained within a common fluidphase during the optically programmable array assembly process. Packetsare dragged-and-dropped so as to maintain an unambiguous correspondencebetween the origin (“reservoir”) of the beads within the packet. At thefinal “drop” position, packets are assembled into subarrays, eachsubarray being composed of a plurality of distinguishable types of“tagged” beads in random positions within the subarray. That is,positions of individual beads are not known a priori. Once at the finallocation, beads within the set can be permanently or temporarilyimmobilized using physical-chemical methods; for examples they can beheld in position using illumination patterns as described herein.

An example of this process is the assembly of arrays of random encodedsubarrays such that heads within each subarray are uniquely identifiedby bead-embedded, in-situ-decodable physical-chemical tags and aplurality of random encoded subarrays are formed in discrete target(“drop”) positions on the substrate surface. Thus, to attain acomplexity of 10,000 types, it surfaces to assemble a 10×10 array ofarrays each containing 100 tag-distinguishable beads. A “randomized”version of this strategy is enabled by sequential injection.

The advantage of this approach is that bead chemistry and substrateprocessing are thereby separated formal the process of forming thearray. For example, different applications such as immunoassays or DNAexpression profiling can be served by the same assembly process, theapplications differing only in the chemical specificity of the beadsemployed. Bead processing including steps such as physical-chemicalencoding as well as surface-attachment of specific chemistries(“functionalization”) as well as quality control may be handled off-lineprior to, array assembly.

Sequential injection, including random sequential injection and beadanchoring, (see FIG. 23) and sequential injection and light-controlledplacement of subarrays (see FIG. 24), may be implemented by connecting aset of individually controllable external reservoirs to the substrate.Alternatively, discrete aliquots of bead suspensions may be depositedonto the substrate (“macro-scale”), with the subsequent assembly ofencoded bead arrays composed of beads extracted from these drops.

FIG. 23 illustrates a method to construct an encoded multi-componenthead array by multiple sequential steps of injection of beadsoriginating in a known reservoir. Each injection step leads, viacollection of particles into the field of view of an imaging instrumentas necessary, to a random configuration of beads: this IS recorded andbeads are immobilized. The next step adds beads to the previousconfiguration. These are highlighted in each new frame. The sequence ofimages, Im_1, Im_2 . . . Im_n provides a record of the configurationsgenerated by each injection step. The array is decoded by looking upindividual images within the sequence and by matching the image obtainedin a binding assay procedure (as previously disclosed herein) with theappropriate image in the sequence.

FIG. 24 illustrates a generalization of the method of FIG. 23.Specifically, a method is illustrated to construct an encodedmulticomponent bead array by multiple steps of injection of beadsoriginating in a known reservoir. Following each injection step, beadsare collected into an illuminated regions and are then moved to adesignated target position via “drag-and-drop” (see FIG. 16) at the“drop” position beads are held stationary or are permanentlyimmobilized. Each “sub-array” can contain a mixture of chemicallyencoded beads.

“Bead Packet Demultiplexing” is achieved in accordance with a preferredembodiment of the present invention, in which sequential injection isimplemented using computer-controlled micro-reservoirs that areconnected by fluidic conduits (“channels”) to ports leading to thesurface of the light-sensitive electrode. Multiple “packets,” of beadsare placed into a continuous field stream and spaced so as to eliminatemutual intermixing between proximal “packets” within the input stream.The sequential order of packets within the stream uniquely representsthe origin of each packet of beads. As each packet emerges on theelectrode surface, each is dragged-and-dropped to a final destination.

This process permits the use of a single input channel for a pluralityof bead types, thereby significantly reducing the complexity of themicrofluidic circuit architecture required to carry beads to thesubstrate surface. This is particularly advantageous when arrayscontaining many distinct subarrays are to be assembled.

An alternative approach is to dispense a suspension containing aplurality of particles from a designated reservoir into a designatedposition on a planar substrate surface in such a way that saidsuspension remains confined in a droplet after deposition and theselected position uniquely identifies each plurality of particles withinthe droplet. T his process may be applied sequentially or concurrentlyto multiple reservoirs and multiple pluralities of particles containedin the reservoirs. Typical volumes of dispensed droplets are 100 nl to 1μl, with adjacent droplets being accordingly spaced so as not to makecontact with their proximal neighbors.

Following deposition of a plurality of droplets onto the bottomelectrode, the top electrode is applied, particle arrays aresimultaneously formed in each droplet, and the electrode gap is closedso as to produce a contiguous fluid phase, Successive “drag-and-drop”operations are applied to pluralities of beads, each such pluralityoriginating in a unique reservoir and each being dropped in a uniquefinal destination on the substrate.

In a specific embodiment, a substrate (“chip”) is deposited in each ofthe wells of a receptacle, the wells being arranged in accordance withthe form factor of standard 8×12 microplates. Multiple bead suspensiondroplets are deposited sequentially on each of the 8×12 chips to produce96 chips carrying arrays of identical composition and layout. Followingdeposition and gap closure, “drag-and drop” operations usingillumination gradients serve to move subarrays into target locationssuch that the target locations of all subarrays on a given chip occupy atotal area in the center of the chip and subarrays are more proximal intheir final positions than in their initial positions (FIGS. 25 a-c).

FIGS. 5 a-c illustrate the combined use of chemical and spatial encodingto enhance the encoding complexity of a particle array. The three panelsaddress related aspects. FIG. 25 a illustrates a method of placingsubstrates (“chips”) into multiple wells. Each chip is configured todisplay a bead array composed of multiple discrete sub-arrays (see FIG.24). By implementing the process of FIG. 24, beads from multiple knownreservoirs are first deposited into droplets on a scale ofdroplet-to-droplet spacings of typically hundreds of microns. Afterdroplets are merged into a contiguous fluid phase, spatially encodedbead arrays are formed by applying a drag-and-drop operation, therebyreducing the spatial scale (subarray-to-subarray spacing) to tens ofmicrons, FIG. 25 b shows the encoding complexities attainable bymultiplying chemical and spatial codes, as discussed herein. FIG. 25 cillustrates the one-to-one correspondence between each subarray (in aknown “drop” position) and the originating reservoir, in the example,reservoirs r1 . . . r8 are shown.

“Banded” assemblies of beads may be formed from a given randomheterogeneous mixture of particles by successively selecting conditionsfavoring the selective assembly of only one specific type of particlepresent in the mixture. These conditions include the optimization ofillumination intensity, and the frequency of the applied electric field.For example, for a given illumination intensity, successively lowerfrequencies favor assembly of successively larger particles. Thesuccessive assembly of particles creates spatially separated “bands”,each such band being composed of only one type of particle and theposition of each such band uniquely identifying the corresponding typeof particle (FIG. 26 a-b).

FIGS. 26 a-b illustrate a method of producing a composite particle arrayexhibiting a concentric set of discrete bands of composition. Thiscomposite structure is produced by performing multiple steps of particleassembly, each step selecting only one type of particle from a randommixture of several types of particles placed on a substrate surface.FIG. 26 a illustrates the process by showing a banded compositecontaining four types of particles denoted by letters A, B, C and D,FIG. 26 b illustrates the realization of such a banded composite arraycontaining an array of 2.8 μm particles: these were assembled underconditions favoring collection of only the smaller particles (see alsoFIGS. 19 and 20 a-b and discussion of characteristic frequencies); thatis, the frequency was chosen such that ω_c (larger particle) <ω<ω_c(smaller particle). In the next step, the frequency was adjusted toω<ω_c (larger particle) to induce the assembly of an array of largerparticles in the shape of a ring surrounding the central array ofsmaller particles. This process can be generalized in the mannerconsidered in FIG. 26 a.

FIG. 27 illustrates the principle of imposing conditions favoringexpulsion of particles from substrate regions illuminated with highintensity under appropriate conditions of voltage and frequency (seediscussion in connection with FIGS. 15 a-b, 19 and 20 a-b), such thatparticles can be subjected to directed “self-assembly” in accordancewith externally imposed layouts. This is illustrated here by aconfiguration of 3.2 μm diameter particles, produced by expulsion ofparticles from a rectangular illuminated region and assembly of theseparticles at a certain distance from the nominal boundaries of theilluminated rectangle in the center of the image. A set of particlesalso decorated the center of the rectangle, In this example, conditionswere similar to those in FIG. 15 b. By invoking intensity gradients inthe form of intersecting profiles particles may be positioned to greatprecision. For example, the intersection of two counterpropagatinglinear profiles (“ramps”) defines a local minimum in the shape of a linealong which particles can line up. This enables the “writing” of linesof particles.

An apparatus according to the present invention may be implemented usingNational Instruments' LabView graphical interface control software on apersonal computer as an operating system to provide the followingfeatures and functions:

-   -   management of all hardware interface and control functions        including input/output modules, image acquisition, digitization        and storage.    -   a graphical programming environment in which to generate code        modules to:        -   generate graphics primitives providing the capability t0            create (“draw”); simple geometric shapes including circle,            ellipse, square and rectangle, composite shapes; and            profiles prescribing a specific intensity variation across a            plurality of pixels;        -   to interactively “drag-and-drop” various shapes using a            graphics input device (“mouse”);        -   to create, store and play back sequences of successive            “drag-and-drop” operations, the sequences of “drop”            positions defining the vertices of a polygonal trajectory,            said vertices being stored in response to “mouse clicks” to            construct and store an entire trajectory; and the play back            speed being interactively adjustable as well as storable            with the trajectory;        -   to load bitmaps containing arbitrarily complex graphical            layouts and designs created off-line using commercially            available graphics or computer aided design software            packages.    -   input for the LCD panel control electronics, with the input        being provided in a standard PC graphics format e.g. VGA, to the        LCD control electronics:    -   a graphical user interface providing interactive management and        programmability of the above functions.

Applications of the method and apparatus of the present inventioninclude the following examples:

High-speed Programmable Particle Array Assembly

Programmable assembly of particles in accordance with complex layouts(“writing”) defining feature sizes via particle size and positioningparticles to submicron precision, for example by invoking gradients ofillumination (FIG. 27).

Creation of “Engineered” Surfaces

Assembly of chemically heterogeneous surfaces in accordance with a giveninjection sequence of multiple pluralities of particle types usingplacement of multiple pluralities of particulates into close packedassemblies in designated areas of the surface. Such surfaces are usefulin a variety of applications, including catalysis.

Non-copyable “Bar Code”

An arrangement, for example in the format of a two-dimensional matrix ofsub-arrays of assembled particles, each composed of a preciselycontrolled number of microparticles, such that the set and coordinatesof occupied positions within the matrix represent a unique code. Usingthis approach, an N×M matrix generates up to 2^(N+M) uniqueconfigurations. FIG. 28 illustrates an example with a 4×4 matrix havingsix fields populated with a random array of beads to produce a unique,miniaturized, non-copyable code. Referring, now to FIG. 28, therein isillustrated a pattern composed of multiple random encoded arrays ofbeads produced by the methods disclosed herein. The pattern represents aunique, miniaturized “label” for the substrate on which it is deposited.To design a unique label, positions within an N×N matrix to be occupiedby bead arrays are randomly chosen. In addition, the position of beadswithin each array is completely random. That is, the structure has twolevels of randomness representing a random matrix of random matriceswhose coding capacity is evaluated in the literature. Replication of thelabel would require the exact, bead by-bead assembly in accordance withthe original structure, a capability that is not required in theoriginal construct ion where only the top level, that is, the placementof entire arrays of beads into designated position, requires spatialcontrol.

Self-tuning Filter/Indicator

A planar array of particles composed so as to partially block incidentlight controlling array assembly. The lateral density of the arrayself-adjusts in accordance with the feedback loop created as follows:

-   -   adjust frequency to a value exceeding the characteristic        dielectric relaxation frequency of constituent particles (this        serves to prevent spontaneous assembly).    -   define illuminated area;    -   adjust illumination to induce collection of particles into, and        assembly within illuminated area;    -   particle assembly within illuminated area will reduce        transmission of light to light-sensitive electrodes thereby        reducing the force attracting particles into the illuminated        area and so reducing particle density;    -   as particle density falls, transmitted intensity rises and        particles are again attracted into illuminated area, with an        optimal density of particles within the illuminated area        emerging.

The advantage of this process is that the optimal lateral density ofparticles within the illuminated area will reflect the selectedillumination intensity and frequency of the applied electric field. Inthis manner, the optimal density serves to “display” the intensity orfrequency, as well as the spatial configuration of the illuminatingsource.

Light-controlled Local Fluid Flows

Induce local fluid flows on the scale of tens to hundreds of microns toinduce micromixing, and lateral transport in accordance with externalillumination patterns. Recirculating flow fields may be created alongthe boundaries of illuminated regions. Also, complex flow patterns areproduced by projecting shapes of desired contours (FIG. 29). Referringnow to FIG. 29, therein is illustrated the light-induced local fluidflow generated at the boundary between illuminated and non-illuminatedregions of the substrate. The recirculating flow field has a toroidalgeometry with inflow along the bottom substrate converging toward theilluminated region and outflow away from the boundary of the region. Theflow velocity increases with voltage and frequency (up to a certainupper limit) of the applied electric field. Complex flow patterns can begenerated by arranging multiple illuminated regions of suitably chosenshape in proximity. This process enables light-induced micromixing andlocal stirring. The example shows a central circular region containing abead array and radially oriented blurry “lines” delineating theperimeter of the region. Close inspection shows that each line pointsupward and away from the illuminated region and is in fact composed ofstrings of particles decorating the recirculating. Particles andoperating conditions in this example were similar to those of FIGS. 15a-b and 19.

Binding Reactions within Random Mixtures. Cross-Linked Bead Clusters

Set up two populations of particles, each population displaying areceptor for one “end” of a long molecule whose two ends are designed tomatch disparate ends of multi-dentate ligand (simplest case: linearmolecule with designed ends: DNA or peptide) that may or may not bepresent in solution. The presence of the ligand is indicated byformation of cross-linked bead clusters.

As an example, two subarrays may be defined, each containing a randommixture of chemically encoded beads, the code corresponding to aspecific receptor displayed on the surface. Mixing is induced (in twodimensions) of two subarrays while recording trajectories for eachparticle in real time. This makes it possible to uniquely distinguish,for example, a red bead originating in subarray 1 (S1) from a red bead(originating in S2: red (S1)!=red (S2); the same Set of colors can so beused for each of the subarrays. Cross-linking may also create dimers (ormore generally, clusters) whose sequence of constituent beads can beanalyzed to reveal the identity of the cross-linked receptors

A Two-dimensional Implementation of Divide-Couple-Recombine Synthesis

Combine capillarity/surface chemical patterning, adjustable gap liquidcell and LEAPS to implement a sequence of reactions typical of DCRstrategy of bead-based solid phase synthesis, as follows:

-   -   form array of fluid droplets each containing a plurality of        particles between parallel electrodes (typically spaced 100 μm        apart) of a liquid cell according to the present invention; each        droplet also is connected to two liquid ports machined into the        two proximal electrodes in matching N×M configurations: ports in        the upper electrode supply aliquots of suspending fluid serving        as the solvent in which each reactive step is carried out; ports        in the lower electrode are equipped with a microporous        “membrane” which serves as a filter permitting solvent to be        suctioned off while retaining beads.

The following sequence of steps is now executed:

-   -   inject suspensions of plurality of particles into N×M positions        via ports in the top electrode;    -   form N×M disjoint droplets (liquid cell gap open)    -   ITERATE        -   add aliquot of N×M reactants in reaction solvent, one per            droplet        -   initiate reaction to add solution-borne reactant to compound            on bead surface        -   add chemical label to head surface to encode reaction        -   form illumination pattern and apply electric field to form            particle array in illuminated areas adjacent to, but not            coincident with, micropores in bottom electrode        -   close gap to form contiguous fluid phase connecting all N×M            droplets        -   increase frequency to disperse particles and use the            principles of the present invention to r-randomly            redistribute particles between positions of an N×M matrix            (via programmed dispersion, segmentation and “drag&drop”            between positions within the N×M array)        -   open gap to reform discrete N×M droplet,        -   suction off fluid (while retaining breads)

END ITERATE

The advantages of this system include the fact that a multiplicity ofparallel on-chip reactions are simultaneously accommodated without theneed to open the planar “reaction chamber” and without the need toremove particles from the chamber for re-arraying between reactionvessels. Reagent consumption is minimized, but more importantly, thecapability is provided to minimize contamination and to handle smallnumbers of beads within a controlled environment which is directlyaccessible to real-time optical monitoring.

While the invention has been particularly shown and described withreference to a preferred embodiment thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention.

1. A process for the manipulation of microparticles, said processcomprising: generating an illumination pattern on a planar surface of asilicon electrode in contact with an electrolyte solution, in whichmicroparticles are suspended; and applying an AC field to said siliconelectrode, whereby said microparticles are transported into or out ofilluminated areas within the illumination pattern.
 2. The process ofclaim 1, wherein the illumination pattern is generated using a graphicaluser environment.
 3. The process of claim 1, wherein said generatingstep is performed by imaging said illumination pattern using a liquidcrystal display panel.
 4. The process of claim 1, wherein theillumination pattern is generated by a generator which includes anactive mask which is configured by means of a video adapter interfacedwith an active mask control circuit.
 5. The process of claim 1, whereinactive feedback is used to programmably reconfigure an array ofmicroparticles assembled on the electrode.
 6. The process of claim 1,wherein said microparticles are segmented into at least two subarrays.